Theramutein modulators

ABSTRACT

This invention relates to agents that are inhibitors or activators of variant forms of endogenous proteins and novel methods of identifying such variants. Of particular interest are inhibitors and activators of endogenous protein variants, encoded by genes which have mutated, which variants often arise or are at least first identified as having arisen following exposure to a chemical agent which is known to be an inhibitor or activator of the corresponding unmutated endogenous protein.

BACKGROUND OF THE INVENTION

The progressive development of drug resistance in a patient is thehallmark of chronic treatment with many classes of drugs, especially inthe therapeutic areas of cancer and infectious diseases. Molecularmechanisms have been identified which mediate certain types of drugresistance phenomena, whereas in other cases the mechanisms of acquiredas well as de novo resistance remain unknown today.

One mechanism of induced (acquired) drug resistance originally thoughtto be relevant in the area of cancer therapy involves increasedexpression of a protein known as P-glycoprotein (P-gp). P-gp is locatedin the cell membrane and functions as a drug efflux pump. The protein iscapable of pumping toxic chemical agents, including many classicalanti-cancer drugs, out of the cell. Consequently, upregulation ofP-glycoprotein usually results in resistance to multiple drugs.Upregulation of P-glycoprotein in tumor cells may represent a defensemechanism which has evolved in mammalian cells to prevent damage fromtoxic chemical agents. Other related drug resistance proteins have nowbeen identified with similar functions to P-gp, includingmultidrug-resistance-associated protein family members such as MRP1 andABCG2. In any event, with the advent of the development of compoundsthat are specific for a given target protein, and less toxic, theimportance of P-glycoprotein and related ATP-binding cassette (ABC)transporter proteins in clinically significant drug resistance haslessened.

Another possible molecular mechanism of acquired drug resistance is thatalternative signal pathways are responsible for continued survival andmetabolism of cells, even though the original drug is still effectiveagainst its target. Furthermore, alterations in intracellular metabolismof the drug can lead to loss of therapeutic efficacy as well. Inaddition, changes in gene expression as well as gene amplificationevents can occur, resulting in increased or decreased expression of agiven target protein, and frequently requiring increasing dosages of thedrug to maintain the same effects. (Adcock and Lane, 2003)

Mutation induced drug resistance is a frequently occurring event in theinfectious disease area. For example, several drugs have been developedthat inhibit either the viral reverse transcriptase or the viralprotease encoded in the human immunodeficiency (HIV) viral genome. It iswell established in the literature that repeated treatment ofHIV-infected AIDS patients using, for example, a reverse transcriptaseinhibitor eventually gives rise to mutant forms of the virus that havereduced sensitivity to the drug which resulted from mutations that haveoccurred in the gene encoding reverse transcriptase that render themutant form of the enzyme less affected by the drug.

The appearance of drug resistance during the course of HIV treatment isnot surprising considering the rate at which errors are introduced intothe HIV genome. The HIV reverse transcriptase enzyme is known to beparticularly error prone, with a forward mutation rate of about 3.4×10⁻⁵mutations per base pair per replication cycle (Mansky et al., J. Virol.69:5087-94 (1995)). However, analogous mutation rates for endogenousgenes encoded in mammalian cells are more than an order of magnitudelower.

New evidence shows that drug resistance can also arise from a mutationalevent involving the gene encoding the drug target (Gorre et al.,Science, 2001; PCT/US02/18729). In this case, exposure of the patient toa specific therapeutic substance such as a given cancer drug thattargets a specific protein-of-interest (POT, or “target” protein) may befollowed by the outgrowth of a group of cells harboring a mutationoccurring in the gene encoding the protein that is the target of thetherapeutic substance. Whether the outgrowth of this population of cellsresults from a small percentage of pre-existing cells in the patientwhich already harbor a mutation which gives rise to a drug-resistantPOI, or whether such mutations arise de novo during or followingexposure of the animal or human being to a therapeutic agent capable ofactivating or inhibiting said POI, is presently unknown. In either case,such mutation events may result in a mutated protein (defined below as atheramutein) which is less affected, or perhaps completely unaffected,by said therapeutic substance.

Chronic myelogenous leukemia (CML) is characterized by excessproliferation of myeloid progenitors that retain the capacity fordifferentiation during the stable or chronic phase of the disease.Multiple lines of evidence have established deregulation of the Abltyrosine kinase as the causative oncogene in certain forms of CML. Thederegulation is commonly associated with a chromosomal translocationknown as the Philadelphia chromosome (Ph), which results in expressionof a fusion protein comprised of the BCR gene product fused to theAbelson tyrosine kinase, thus forming p210^(Bcr-Abl) which has tyrosinekinase activity. A related fusion protein, termed p190^(Bcr-Abl), thatarises from a different breakpoint in the BCR gene, and has been shownto occur in patients with Philadelphia chromosome positive (Ph+) AcuteLymphoblastic Leukemia (ALL) (Melo, 1994; Ravandi et al., 1999).Transformation appears to result from activation of multiple signalpathways including those involving RAS, MYC, and JUN. Imatinib mesylate(“STI-571” or “Gleevec®”) is a 2-phenylamino pyrimidine that targets theATP binding site of the kinase domain of Abl (Druker et al, NEJM 2001,p. 1038). Subsequently it has also been found by other methods to be aninhibitor of platelet-derived growth factor (PDGF) β receptor, and theKit tyrosine kinase, the latter of which is involved in the developmentof gastrointestinal stromal tumors (see below).

Until recently, it had not been observed that during the course oftreatment with a specific inhibitor of a given endogenous cellularprotein that a mutation in its corresponding endogenous gene could leadto the expression of protein variants whose cellular functioning wasresistant to the inhibitor. Work by Charles Sawyers and colleagues(Gorre et al., Science 293:876-80 (2001); PCT/US02/18729) demonstratedfor the first time that treatment of a patient with a drug capable ofinhibiting the p210^(Bcr-Abl) tyrosine kinase (i.e., STI-571) could befollowed by the emergence of a clinically significant population ofcells within said patient harboring a mutation in the gene encoding thep210^(Bcr-Abl) cancer causing target protein which contains the Abelsontyrosine kinase domain. Various such mutations gave rise to mutant formsof p210^(Bcr-Abl) which were less responsive to Gleevec treatment thanwas the original cancer causing version. Notably, the mutations thatemerged conferred upon the mutant protein a relative resistance to theeffects of the protein kinase inhibitor drug, while maintaining acertain degree of the original substrate specificity of the mutantprotein kinase. Prior to Gorre et al.'s work, it was generally believedby those skilled in the art that the types of resistance that would beobserved in patients exposed to a compound which inhibited the Abelsonprotein kinase, such as STI-571, would have resulted from one or more ofthe other mechanisms of drug resistance listed above, or by some otheras yet unknown mechanism, but that in any event said resistance wouldinvolve a target (protein or otherwise) which was distinct from thedrug's target POI.

Accordingly, the ability to treat clinically relevant resistant mutantforms of proteins that are otherwise the targets of an existing therapywould be extremely useful. Such mutated proteins (theramuteins asdefined below) are beginning to be recognized and understood to beimportant targets in recurring cancers, and will become important inother diseases as well. There exists a need for therapeutic agents thatare active against such drug resistant variant forms of cellularproteins that may arise before, during or following normally effectivedrug therapies. A key purpose of this invention is to provide compoundsthat may serve as potential therapeutic agents useful in overcomingmutation-induced drug resistance in endogenously occurring proteins.

BRIEF SUMMARY OF THE INVENTION

This invention relates to agents that are inhibitors or activators ofvariant forms of endogenous proteins and novel methods of identifyingsuch variants. Of particular interest are inhibitors and activators ofendogenous protein variants, encoded by genes which have mutated, whichvariants often arise or are at least first identified as having arisenfollowing exposure to a chemical agent which is known to be an inhibitoror activator of the corresponding unmutated endogenous protein. Suchprotein variants (mutant proteins) are herein termed “theramuteins,” mayoccur either spontaneously in an organism (and be pre-existing mutationsin some cases), or said mutants may arise as a result of selectivepressure which results when the organism is treated with a givenchemical agent capable of inhibiting the non-mutated form of saidtheramutein (herein termed a “prototheramutein”). It will be understoodthat in some cases a prototheramutein may be a “wild type” form of a POI(e.g., a protein that gives rise to a disease due to disregulation). Inother cases, the prototheramutein will be a disease causing variant of a“wild type” protein, having already mutated and thereby contributing tothe development of the diseased state as a result of said priormutation. One example of the latter type of prototheramutein is theP210^(BCR-ABL) oncoprotein, and a mutant form of this protein harboringa threonine (T) to isoleucine (I) mutation at position 315 is termedP210^(BCR-ABL-T315I) and is one example of a theramutein. As usedherein, the designation “P210^(BCR-ABL)” is synonymous with the term“p210^(Bcr-Abl)”, the “wild-type Bcr-Abl protein”, and the like.

Theramuteins are a rare class of endogenous proteins that harbormutations that render said proteins resistant to drugs that are known toinhibit or activate in a therapeutically effective manner theirnon-mutated counterparts. The endogenous genes encoding a few suchproteins are presently known to exhibit such mutations under certaincircumstances. This Invention is directed toward compositions thatinhibit certain drug-resistant mutants (theramuteins) of the Abelsontyrosine kinase protein, originally termed P210-Bcr-Abl in theliterature, that is involved in the development of chronic myelogenousleukemia. The invention is also directed toward general methods ofidentifying compounds that inhibit or activate any theramutein.

The present method is particularly directed toward the identification ofspecific inhibitors or specific activators of theramuteins. Use of theterm “specific” in the context of the terms “inhibitor” or “activator”(see definitions below) means that said inhibitor or activator binds tothe theramutein and inhibits or activates the cellular functioning ofthe theramutein without also binding to and activating or inhibiting awide variety of other proteins or non-protein targets in the cell. Theskilled investigator is well aware that there is a certain degree ofvariability in the medical literature with respect to the concept of aspecific inhibitor or a specific activator, and of the related conceptof target protein “specificity” when discussing the actions ofinhibitors or activators of a protein. Accordingly, for the purposes ofthis Invention, a substance is a specific inhibitor or a specificactivator of a given theramutein if said substance is capable ofinhibiting or activating said theramutein at a given concentration suchthat a corresponding phenoresponse is modulated in the appropriatemanner, without having an appreciable effect at the same givenconcentration upon the phenoresponse of a corresponding control cellthat essentially does not express either the theramutein or itscorresponding prototheramutein.

In certain embodiments, a substance may be a modulator of theprototheramutein as well as the theramutein. In other embodiments, inaddition to being a modulator of the prototheramutein and theramutein, asubstance may also modulate the activities of proteins that have similarfunctions. As discussed above, in addition to inhibiting thep210^(Bcr Abl) tyrosine kinase, imatinib mesylate is also capable ofinhibiting the c-kit oncogene product (also a tyrosine kinase) which isoverexpressed in certain gastrointestinal stromal tumors, as well as thePDGF R receptor (also a tyrosine kinase), which is expressed in certainchronic myelomonocytic leukemias (CMML). Such a compound is sometimestermed a “moderately specific” inhibitor.

The invention also provides a general method that can be used toidentify substances that will activate or inhibit a theramutein, to thesame extent, and preferably to an even greater extent than a known drugsubstance is capable of inhibiting the corresponding “wild type” form ofthat protein. (The skilled artisan is well aware, however, that said“wild type” forms of such proteins may have already mutated in thecourse of giving rise to the corresponding disease in which said proteinparticipates.)

In a preferred embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula I

wherein:

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹;-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   R² is selected from —CR²¹ _(a)—, —NR²² _(b)—, and —(C═R²³)—;    -   each R²¹ is independently selected from H, halo, —NH₂,        —N(H)(C₁₋₃ alkyl), —N(C₁₋₃ alkyl)₂, —O—(C₁₋₃ alkyl), OH and C₁₋₃        alkyl;    -   each R²² is independently selected from H and C₁₋₃ alkyl;    -   R²³ is selected from O, S, N—R⁰, and N—OR⁰;-   R³ is selected from —CR³¹ _(c)—, —NR³² _(d)—, and —(C═R³³)—;    -   each R³¹ group is selected from H, halo, —NH₂, —N(H)(R⁰),        —N(R⁰)₂, —O—R⁰, OH and C₁₋₃ alkyl;    -   each R³² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    -   R³³ is selected from O, S, N—R³⁴, and N—OR⁰;    -   R³⁴ is selected from H, NO₂, CN, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, aryl and a heterocyclic ring;-   R⁴ is selected from —CR⁴¹ _(e)—, —NR⁴² _(f)—, —(C═R⁴³)—, and —O—;    -   each R⁴¹ is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl, and a heterocyclic ring;    -   each R⁴² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    -   each R⁴³ is selected from O, S, N—R⁰, and N—OR⁰;-   with the provisos that when R² is —NR²² _(b)— and R⁴ is —NR⁴² _(f)—,    then R³ is not —NR³² _(d)—; and that both R³ and R⁴ are not    simultaneously selected from —(C═R³³)— and —(C═R⁴³)—, respectively;-   R⁵ is selected from —Y—R⁶ and —Z—R⁷;    -   Y is selected from a chemical bond, O, NR⁰,    -   R⁶ is selected from alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and        optionally substituted with one or more of halo, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R⁰, C(O)R⁰,        C(O)N(R⁵)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;    -   R⁷ is H or is selected from aryl and a heterocyclic ring;-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;-   a is 1 or 2;-   b is 0 or 1;-   c is 1 or 2;-   d is 0 or 1;-   e is 1 or 2; and-   f is 0 or 1.

The invention provides for a fundamentally new way of treating cancerand other diseases where treatment with an existing drug compound, bywhatever mechanism, is followed by identifiable (clinically significant)theramutein-mediated drug resistance, by providing alternative drugsthat can be administered as theramuteins arise and are identified assuch (Wakai et al., 2004, reports an example wherein a theramutein mayarise during the course of an on-going treatment regimen), orpreemptively before the outgrowth of clinically significant populationsof theramutein expressing cells. Further, where a drug treatment for aparticular disease is less effective in a subset of individuals thatexpress a certain theramutein of a protein that the drug targets, theinvention enables the tailoring of treatments for those subjects byproviding alternative drug substances that will be effective againstsaid theramutein.

1. The invention provides a method of determining whether a chemicalagent is at least as effective a modulator of a theramutein in a cell asa known substance is a modulator of a corresponding prototheramutein.One embodiment of the method involves contacting a control cell thatexpresses the prototheramutein and is capable of exhibiting a responsivephenotypic characteristic (linked to the functioning of theprototheramutein in the cell) with the known modulator of theprototheramutein, contacting a test cell that expresses the theramuteinand is also capable of exhibiting the responsive phenotypiccharacteristic (linked to the functioning of the theramutein in thecell) with the chemical agent, and comparing the response of the treatedtest cell with the response of the treated control cell; to determinethat the chemical agent is at least as effective a modulator of thetheramutein as the known substance is a modulator of theprototheramutein. In certain other embodiments, one type of control cellmay not express the prototheramutein at all. In other embodiments, thecontrol cell may express about the same amount of the prototheramuteinas the test cell expresses of the theramutein. In still otherembodiments, the control cell may be capable of exhibiting theresponsive phenotypic characteristic to about the same extent as thetest cell under certain conditions.

2. Theramuteins of the invention that are of particular interest arethose involved in regulatory function, such as enzymes, protein kinases,tyrosine kinases, receptor tyrosine kinases, serine threonine proteinkinases, dual specificity protein kinases, proteases, matrixmetalloproteinases, phosphatases, cell cycle control proteins, dockingproteins such as the IRS family members, cell-surface receptors,G-proteins, ion channels, DNA- and RNA-binding proteins, polymerases,and the like. No limitation is intended on the type of theramutein thatmay be used in the invention. At the present time, three theramuteinsare known: BCR-ABL, c-Kit, and EGFR.

3. Any responsive phenotypic characteristic that can be linked to thepresence of the theramutein (or prototheramutein) in the cell can beemployed for use in the method, including, for example, growth orculture properties, the phosphorylation state (or other modification) ofa substrate of the theramutein, and any type of transient characteristicof the cell, as will be defined and discussed in detail

DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect on growth and viability of differentconcentrations of Compound 2 (C2) for non-transformed vector controlBa/F3 cells (which are IL-3 dependent) as well as Ba/F3 cells expressingthe “wild type” p210^(Bcr-Abl) (designated p210^(Bcr-Abl-wt)), and Ba/F3cells expressing the p210^(Bcr-Abl-T315I) drug resistant mutant. Cellcounts and viability were determined on an automated cell counter asdiscussed in detail in the specification. Cell counts are shown by thesolid color bars; cell viability is shown by the hashed bars. Note thatSTI-571 potently inhibits growth of the P210 cell line (grey bar)whereas it is unable to inhibit the growth of the T315I cell line (whitebar) even at 10 μM concentration. 500 nM C2 shows the largestspecificity gap within this dose-response series. Compare STI-571 at 10μM to C2 at 500 nM on the T315I cell line (white bars). Abbreviations:DMSO: dimethylsulfoxide (solvent used for drug dissolution).

FIG. 2 shows the effect on growth and viability of differentconcentrations of Compound 6 (C6) for non-transformed vector controlBa/F3 cells as well as Ba/F3 cells expressing the p210^(Bcr-Abl-T315I)drug resistant mutant. All other details are as per FIG. 1.

FIG. 3 shows various determinations of the specificity gap by comparingthe effects of various compounds identified in the screen in terms oftheir effects on the prototheramutein- and theramutein-expressing celllines. Compound 3 (C3) shows the best example of the ability of themethod to identify a compound that exerts an even greater effect on thetheramutein than on its corresponding prototheramutein. (Panel E). PanelA: control DMSO treatments; B: negative heterologous specificity gap; C:slightly positive heterologous specificity gap; D: large positivehomologous specificity gap; E: positive heterologous specificity gap.See text for explanations.

FIG. 4 shows an autoradiograph of recombinant P210 Bcr-Abl wild type andT315I mutant kinase domains assayed for autophosphorylation activity.200 ng of protein were preincubated with test substances for 10 minutesunder standard autophosphoryation reaction conditions and thenradiolabelled ATP was added and the reactions proceeded for 30 minutesat 30° C., after which the samples were separated by SDS-PAGE. The gelswere silver-stained, dried down under vacuum and exposed to X-ray film.Note that whereas 10 μM STI 571 is effective against wild type P210Bcr-Abl, it is virtually ineffective against the T315I kinase domain,even at concentrations up to 100 μM. C2 and C6 are the best twocompounds identified, followed by C5, C7 and C4. All of the compoundstested positively to some extent. “P210 cell line” refers to cellsexpressing p210^(BCR-ABL-wt). “T315I cell line” refers to cellsexpressing p210^(BCR-ABL-T315I).

FIG. 5 shows the chemical structures of representative compounds of thepresent invention.

FIG. 6 shows the chemical structures of representative compounds of thepresent invention.

FIG. 7 shows the chemical structures of representative compounds of thepresent invention.

FIG. 8 shows the chemical structures of representative compounds of thepresent invention.

FIG. 9 shows the chemical structures of representative compounds of thepresent invention.

FIG. 10 shows the chemical structures of representative compounds of thepresent invention.

FIG. 11 shows the chemical structures of representative compounds of thepresent invention.

FIG. 12 shows the chemical structures of representative compounds of thepresent invention.

FIG. 13 shows the chemical structures of representative compounds of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The term “halo” or “halogen” as used herein includes fluorine, chlorine,bromine and iodine.

The term “alkyl” as used herein contemplates substituted andunsubstituted, straight and branched chain alkyl radicals having from 1to 6 carbon atoms. Preferred alkyl groups include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, and the like. Additionally, thealkyl group may be optionally substituted with one or more substituentsselected from halo, CN, CO₂R, C(O)R, C(O)NR₂, NR₂, cyclic-amino, NO₂,and OR.

The term “cycloalkyl” as used herein contemplates substituted andunsubstituted cyclic alkyl radicals. Preferred cycloalkyl groups arethose with a single ring containing 3 to 7 carbon atoms and includescyclopropyl, cyclopentyl, cyclohexyl, and the like. Other cycloalkylgroups may be selected from C₇ to C₁₀ bicyclic systems or from C₉ to C₁₄tricyclic systems. Additionally, the cycloalkyl group may be optionallysubstituted with one or more substituents selected from halo, CN, CO₂R,C(O)R, C(O)NR₂, NR₂, cyclic-amino, NO₂, and OR.

The term “alkenyl” as used herein contemplates substituted andunsubstituted, straight and branched chain alkene radicals. Preferredalkenyl groups are those containing two to six carbon atoms.Additionally, the alkenyl group may be optionally substituted with oneor more substituents selected from halo, CN, CO₂R, C(O)R, C(O)NR₂, NR₂,cyclic-amino, NO₂, and OR.

The term “alkynyl” as used herein contemplates substituted andunsubstituted, straight and branched chain alkyne radicals. Preferredalkynyl groups are those containing two to six carbon atoms.Additionally, the alkynyl group may be optionally substituted with oneor more substituents selected from halo, CN, CO₂R, C(O)R, C(O)NR₂, NR₂,cyclic-amino, NO₂, and OR.

The term “aralkyl” as used herein contemplates an alkyl group which hasas a substituent an aromatic group, which aromatic group may besubstituted and unsubstituted. The aralkyl group may be optionallysubstituted on the aryl with one or more substituents selected fromhalo, CN, CF₃, NR₂, cyclic-amino, NO₂, OR, CF₃,—(CH₂)_(x)C(O)(CH₂)_(y)R, —(CH₂)_(x)C(O)N(R′)(R″),—(CH₂)_(x)C(O)O(CH₂)_(y)R, —(CH₂)_(x)N(R′)(R″), —N(R)SO₂R,—O(CH₂)_(x)C(O)N(R′)(R″), —SO₂N(R′)(R″), —(CH₂)_(x)N(R)—(CH₂)_(y)—R,—(CH₂)_(x)N(R)—C(O)—(CH₂)_(y)—R, —(CH₂)_(x)N(R)—C(O)—O—(CH₂)_(y)—R,—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, —(CH₂)_(x)C(O)N(R)—(CH₂)_(y)—R,—O—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, substituted and unsubstituted alkyl,substituted and unsubstituted cycloalkyl, substituted and unsubstitutedaralkyl, substituted and unsubstituted alkenyl, substituted andunsubstituted alkynyl, substituted and unsubstituted aryl, and asubstituted and unsubstituted heterocyclic ring, wherein the substitutedalkyl, substituted cycloalkyl, substituted aralkyl, substituted alkenyl,substituted alkynyl, substituted aryl, and substituted heterocyclic ringmay be substituted with one of more halo, CN, CF₃, CO₂R, C(O)R, C(O)NR₂,NR₂, cyclic-amino, NO₂, and OR.

The term “heterocyclic group” or “heterocyclic ring” as used hereincontemplates aromatic and non-aromatic cyclic radicals having at leastone heteroatom as a ring member. Preferred heterocyclic groups are thosecontaining 5 or 6 ring atoms which includes at least one hetero atom,and includes cyclic amines such as morpholino, piperidino, pyrrolidino,and the like, and cyclic ethers, such as tetrahydrofuran,tetrahydropyran, and the like. Aromatic heterocyclic groups, also termed“heteroaryl” groups contemplates single-ring hetero-aromatic groups thatmay include from one to three heteroatoms, for example, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,pyrazine, pyridazine, pyrimidine, and the like. The term heteroaryl alsoincludes polycyclic hetero-aromatic systems having two or more rings inwhich two atoms are common to two adjoining rings (the rings are“fused”) wherein at least one of the rings is a heteroaryl, e.g., theother rings can be cycloalkyls, cycloalkenyls, aryl, heterocycles and/orheteroaryls. Examples of polycyclic heteroaromatic systems includequinoline, isoquinoline, tetrahydroisoquinoline, quinoxaline,quinaxoline, benzimidazole, benzofuran, purine, imidazopyridine,benzotriazole, and the like. Additionally, the heterocyclic groups maybe optionally substituted with halo, CN, CF₃, NR₂, cyclic-amino, NO₂,OR, CF₃, —(CH₂)_(x)C(O)(CH₂)_(y)R, —(CH₂)_(x)C(O)N(R′)(R″),—(CH₂)_(x)C(O)O(CH₂)_(y)R, —(CH₂)_(x)N(R′)(R″), —N(R)SO₂R,—O(CH₂)_(x)C(O)N(R′)(R″), —SO₂N(R′)(R″), —(CH₂)_(x)N(R)—(CH₂)_(y)—R,—(CH₂)_(x)N(R)—C(O)—(CH₂)_(y)—R, —(CH₂)_(x)N(R)—C(O)—O—(CH₂)_(y)—R,—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, —(CH₂)_(x)C(O)N(R)—(CH₂)_(y)—R,—O—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, substituted and unsubstituted alkyl,substituted and unsubstituted cycloalkyl, substituted and unsubstitutedaralkyl, substituted and unsubstituted alkenyl, substituted andunsubstituted alkynyl, substituted and unsubstituted aryl, and asubstituted and unsubstituted heterocyclic ring, wherein the substitutedalkyl, substituted cycloalkyl, substituted aralkyl, substituted alkenyl,substituted alkynyl, substituted aryl, and substituted heterocyclic ringmay be substituted with one of more halo, CN, CF₃, CO₂R, C(O)R, C(O)NR₂,NR₂, cyclic-amino, NO₂, and OR.

The term “aryl” or “aromatic group” as used herein contemplatessingle-ring aromatic groups (for example, phenyl, pyridyl, pyrazole,etc.) and polycyclic ring systems (naphthyl, quinoline, etc.). Thepolycyclic rings may have two or more rings in which two atoms arecommon to two adjoining rings (the rings are “fused”) wherein at leastone of the rings is aromatic, e.g., the other rings can be cycloalkyls,cycloalkenyls, aryl, heterocycles and/or heteroaryls. Additionally, thearyl groups may be optionally substituted with one or more substituentsselected from halo, CN, CF₃, NR₂, cyclic-amino, NO₂, OR, CF₃,—(CH₂)_(x)C(O)(CH₂)_(y)R, —(CH₂)_(x)C(O)N(R′)(R″),—(CH₂)_(x)C(O)O(CH₂)_(y)R, —(CH₂)_(x)N(R′)(R″), —N(R)SO₂R,—O(CH₂)_(x)C(O)N(R′)(R″), —SO₂N(R′)(R″), —(CH₂)_(x)N(R)—(CH₂)_(y)—R,—(CH₂)_(x)N(R)—C(O)—(CH₂)_(y)—R, —(CH₂)_(x)N(R)—C(O)—O—(CH₂)_(y)—R,—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, —(CH₂)_(x)C(O)N(R)—(CH₂)_(y)—R,—O—(CH₂)_(x)—C(O)—N(R)—(CH₂)_(y)—R, substituted and unsubstituted alkyl,substituted and unsubstituted cycloalkyl, substituted and unsubstitutedaralkyl, substituted and unsubstituted alkenyl, substituted andunsubstituted alkynyl, substituted and unsubstituted aryl, and asubstituted and unsubstituted heterocyclic ring, wherein the substitutedalkyl, substituted cycloalkyl, substituted aralkyl, substituted alkenyl,substituted alkynyl, substituted aryl, and substituted heterocyclic ringmay be substituted with one of more halo, CN, CF₃, CO₂R, C(O)R, C(O)NR₂,NR₂, cyclic-amino, NO₂, and OR.

The term “heteroatom”, particularly as a ring heteroatom, refers to N,O, and S.

Each R is independently selected from H, substituted and unsubstitutedalkyl, substituted and unsubstituted cycloalkyl, substituted andunsubstituted aralkyl, substituted and unsubstituted aryl and asubstituted and unsubstituted heterocyclic ring, wherein the substitutedalkyl, substituted cycloalkyl, substituted aralkyl, substituted aryl andsubstituted heterocyclic ring may be substituted with one or more halo,CN, CF₃, OH, CO₂H, NO₂, C₁₋₆alkyl, —O—(C₁₋₆alkyl), —NH₂, —NH(C₁₋₆alkyl)and —N(C₁₋₆alkyl)₂. Each R′ and R″ are independently selected from H, orsubstituted and unsubstituted alkyl, substituted and unsubstitutedcycloalkyl, substituted and unsubstituted aralkyl, substituted andunsubstituted aryl and a substituted and unsubstituted heterocyclicring, wherein the substituted alkyl, substituted cycloalkyl, substitutedaralkyl, substituted aryl and substituted heterocyclic ring may besubstituted with one or more halo, CN, CF₃, OH, CO₂H, NO₂, C₁₋₆alkyl,—O—(C₁₋₆alkyl), —NH₂, —NH(C₁₋₆alkyl) and —N(C₁₋₆alkyl)₂; or R′ and R″may be taken together with the nitrogen to which they are attached forma 5- to 7-membered ring which may optionally contain up to three furtherheteroatoms. Each x and each y are independently selected from 0 to 4.

In a preferred embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula I

wherein:

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹.-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   R² is selected from —CR²¹ _(a)—, —NR²² _(b)—, and —(C═R²³)—;    -   each R²¹ is independently selected from H, halo, —NH₂,        —N(H)(C₁₋₃ alkyl), —N(C₁₋₃ alkyl)₂, —O—(C₁₋₃ alkyl), OH and C₁₋₃        alkyl;    -   each R²² is independently selected from H and C₁₋₃ alkyl;    -   R²³ is selected from O, S, N—R⁰, and N—OR⁰;-   R³ is selected from —CR³¹ _(c)—, —NR³² _(d)—, and —(C═R³³)—;    -   each R³¹ group is selected from H, halo, —NH₂, —N(H)(R⁰),        —N(R⁰)₂, —O—R⁰, OH and C₁₋₃ alkyl;    -   each R³² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    -   R³³ is selected from O, S, N—R³⁴, and N—OR⁰;    -   R³⁴ is selected from H, NO₂, CN, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, aryl and a heterocyclic ring;-   R⁴ is selected from —CR⁴¹ _(e)—, —NR⁴² _(f)—, —(C═R⁴³)—, and —O—;    -   each R⁴¹ is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl, and a heterocyclic ring;    -   each R⁴² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    -   each R⁴³ is selected from O, S, N—R⁰, and N—OR⁰;-   with the provisos that when R² is —NR²² _(b)— and R⁴ is —NR⁴² _(f)—,    then R³ is not —NR³² _(d)—; and that both R³ and R⁴ are not    simultaneously selected from —(C═R³³)— and —(C═R⁴³)—, respectively;-   R⁵ is selected from —Y—R⁶ and —Z—R⁷;    -   Y is selected from a chemical bond, O, NR⁰,    -   R⁶ is selected from alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and        optionally substituted with one or more of halo, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R⁰, C(O)R⁰,        C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;    -   R⁷ is H or is selected from aryl and a heterocyclic ring;-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;-   a is 1 or 2;-   b is 0 or 1;-   c is 1 or 2;-   d is 0 or 1;-   e is 1 or 2; and-   f is 0 or 1.

An important component and conceptual teaching of the Inventiondescribed herein is that neither the R² nor the R³ positions of thecompounds of this invention are members of any aromatic or non-aromaticring structure. We have discovered that compounds having the R² and/orthe R³ positions as members of any aromatic or non-aromatic ringstructure do not effectively inhibit the T315I theramutein, whereas thecompounds of the invention that lack such a ring component at thesepositions, in addition to having other preferred chemical groups, arepotent inhibitors of the T315I theramutein.

In preferred embodiments of the invention, ring A is an aromatic ring.

In preferred embodiments of the invention, X¹ or X² is N. In anotherpreferred embodiment, both X¹ and X² are N. In particularly preferredembodiments of the invention Ring A is a pyridine ring or a pyrimidinering. In still further preferred embodiments, Ring A is selected fromthe structures provided below:

In a preferred embodiment, if R² or R⁴ is selected to be —NR²² _(b)— or—NR⁴²—, respectively, then R³¹ is not selected from halo, —NH₂,—N(H)(R⁰), —N(R⁰)₂, —O—R⁰, or OH.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaI_(a)

wherein:

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹;-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   each R² is independently selected from H and C₁₋₃ alkyl;-   R³ is selected from —CR³¹ _(c)—, —NR³² _(d)—, and —(C═R³³)—;    -   each R³¹ group is selected from H, halo, —NH₂, —N(H)(R⁰),        —N(R⁰)₂, —O—R⁰, OH and C₁₋₃ alkyl;    -   each R³² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    -   R³³ is selected from O, S, N—R³⁴, and N—OR⁰;    -   R³⁴ is selected from H, NO₂, CN, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, aryl and a heterocyclic ring;-   R⁴ is selected from —CR⁴¹ _(e)—, —NR⁴² _(f)—, —(C═R⁴³)—, and —O—;    -   each R⁴¹ is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl, and a heterocyclic ring;    -   each R⁴² group is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;    -   each R⁴³ is selected from O, S, N—R⁰, and N—OR⁰;-   with the provisos that when R⁴ is —NR⁴² _(f)—, then R³ is not —NR³²    _(d)—; and that both R³ and R⁴ are not simultaneously selected from    —(C═R³³)— and —(C═R⁴³)—, respectively;-   R⁵ is selected from —Y—R⁶ and —Z—R⁷;    -   Y is selected from a chemical bond, O, N—R⁰,    -   R⁶ is selected from alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and        optionally substituted with one or more of halo, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R⁰, C(O)R⁰,        C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;    -   R⁷ is H or is selected from aryl and a heterocyclic ring;-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;-   a is 1 or 2;-   h is 0 or 1;-   c is 1 or 2;-   d is 0 or 1;-   e is 1 or 2; and-   f is 0 or 1.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaI_(b)

wherein:

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹;-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   each R²² is independently selected from H and C₁₋₃ alkyl;-   each R³² group is selected from H, alkyl, cycloalkyl, alkenyl,    alkynyl, aralkyl, CO₂R⁰, C(O)R⁰, aryl, and a heterocyclic ring;-   R⁴ is selected from —CR⁴¹ _(e)—, —(C═R⁴³)—, and —O—;    -   each R⁴¹ is selected from H, alkyl, cycloalkyl, alkenyl,        alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl, and a heterocyclic ring;    -   each R⁴³ is selected from O, S, N—R⁰, and N—OR⁰;-   R⁵ is selected from —Y—R⁶ and —Z—R⁰;    -   Y is selected from a chemical bond, O, N—R⁰,    -   R⁶ is selected from alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and        optionally substituted with one or more of halo, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R⁰, C(O)R⁰,        C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;    -   R⁷ is H or is selected from aryl and a heterocyclic ring;-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;-   a is 1 or 2;-   b is 0 or 1;-   c is 1 or 2;-   d is 0 or 1;-   e is 1 or 2; and-   f is 0 or 1.

In preferred embodiments of the invention, R², R³ and R⁴ of formula Iare selected to give the following chemical groups:

-   -   —N(R²²)—N═C(R⁴¹)—    -   —N(R²²)—N(R³²)—C(═O)—    -   —N(R²²)—N(R³²)—C(R⁴¹)(R⁴¹)—    -   —N(R²²)—C(R³¹)(R³¹)—C(R⁴¹)(R⁴¹)—    -   —N(R²²)—C(R³¹)(R³¹)—C(═O)—    -   —N═N—C(R⁴¹)(R⁴¹)—    -   —C(R²¹)═C═C(R⁴¹)—    -   —C(R²¹)═C(R³¹)—C(═O)—    -   —C(R²¹)═C(R³¹)—C(R⁴¹)(R⁴¹)—    -   —C(R²¹)(R²¹)—C(R³¹)═C(R⁴¹)—    -   —C(R²¹)(R²¹)—C(R³¹)(R³¹)—C(═O)—    -   —C(R²¹)(R²¹)—C(R³¹)(R³¹)—C(R⁴¹)(R⁴¹)—    -   —C(R²¹)(R²¹)—N(R³²)—C(═O)—    -   —C(R²¹)(R²¹)—N(R³²)—C(R⁴¹)(R⁴¹)—    -   —N(R²²)—C(═O)—C(R⁴¹)(R⁴¹)—    -   —N(R²²)—C(═O)—N(R⁴¹)—    -   —N(R²²)—C(═O)—O—    -   —C(R²¹)(R²¹)—C(═O)—C(R⁴¹)(R⁴¹)—    -   —C(R²¹)(R²¹)—C(═O)—N(R⁴²)—    -   —N(R²²)—C(═NR³⁴)—N(R⁴²)—    -   —C(═O)—N(R³²)—N(R⁴²).        Particularly preferred chemical groups for R², R³ and R⁴        include:    -   —N(R²²)—N═C(R⁴¹)—    -   —N(R²²)—N(R³²)—C(═O)—    -   —N(R²²)—C(R³¹)(R³¹)—C(R⁴¹)(R⁴¹)—    -   —N(R²²)—C(R³¹)(R³¹)—C(═O)—    -   —C(R²¹)(R²¹)—C(═O)—C(R⁴¹)(R⁴¹)—    -   —C(R²¹)(R²¹)—C(═O)—N(R⁴²)—    -   —N(R²²)—C(═NR³⁴)—N(R⁴²)—    -   —C(═O)—N(R³²)—N(R⁴²).

In further preferred embodiment, R⁶ or R⁷ is an aryl group, which may beoptionally substituted. Particularly preferred aryl groups includesubstituted or unsubstituted phenyl and pyridyl. In additional oralternative embodiments, it is preferred that the substituents R²¹ andR²² are independently selected from groups which have small steric bulkand are preferably selected from H and CH₃, and more preferably are H.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formula II

wherein

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹;-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   R⁸ is selected from the group consisting of is selected from H,    alkyl, cycloalkyl, alkenyl, alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl,    and a heterocyclic ring;-   R⁹ is selected from —Y—R⁶ and —Z—R⁷;    -   Y is selected from a chemical bond, O, N—R⁰,    -   R⁶ is selected from alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   Z is a hydrocarbon chain having from 1 to 4 carbon atoms, and        optionally substituted with one or more of halo, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R⁰, C(O)R⁰,        C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;    -   R⁷ is H or is selected from aryl and a heterocyclic ring; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaII_(a)

wherein

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹;-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   R⁸ is selected from the group consisting of is selected from H,    alkyl, cycloalkyl, alkenyl, alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl,    and a heterocyclic ring; X³ is N, CH or C—R⁵⁰;-   each R⁵⁰ is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(r)C(O)(CH₂)_(s)R⁵¹, —(CH₂)_(r)C(O)N(R⁵²)(R⁵³),    —(CH₂)_(r)C(O)O(CH₂)_(s)R⁵¹, —(CH₂)_(r)N(R⁵¹)C(O)R⁵¹,    —(CH₂)_(r)N(R⁵²)(R⁵³), —N(R⁵¹)SO₂R⁵¹, —OC(O)N(R⁵²)(R⁵³),    —SO₂N(R⁵²)(R⁵³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R⁵⁰ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    -   R⁵¹ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   R⁵² and R⁵³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R⁵² and R⁵³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   r is 0 to 4;    -   s is 0 to 4;-   m is 0 to 4; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(CR-ABL-T315I) theramutein having the formulaII_(b)

wherein:

-   R¹⁴ is selected from H and F;-   R⁸ is selected from the group consisting of is selected from H,    alkyl, cycloalkyl, alkenyl, alkynyl, CO₂R⁰, C(O)R⁰, aralkyl, aryl,    and a heterocyclic ring;-   X³ is N, CH or C—R⁶⁰;-   each R⁶⁰ is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁰,    halo, aryl, and a heterocyclic ring;-   R⁶¹ is selected from aryl and a heterocyclic ring;-   Q is selected from a chemical bond or a group having the formula    —O—, —(CH₂)_(i)—, —(CH₂)_(i)C(O)(CH₂)_(j)—,    —(CH₂)_(i)—N(R⁶²)—(CH₂)_(j)—, —(CH₂)_(i)C(O)—N(R⁶²)—(CH₂)_(j)—,    —(CH₂)_(i)C(O)O(CH₂)_(j)—, —(CH₂)_(i)N(R⁶²)C(O)—(CH₂)_(j)—,    —(CH₂)_(i)OC(O)N(R⁶²)—(CH₂)_(j)—, and    —O—(CH₂)_(i)—C(O)N(R⁶²)—(CH₂)_(j)—;-   R⁶² is selected from aryl, and a heterocyclic ring;-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring;-   h is 0 to 4;-   i is 0 to 4; and-   j is 0 to 4.

In preferred embodiments of compounds of the formula II_(b), R⁶⁰ isselected from halo, CF₃, and OH.

Exemplary compounds of the formula II, II_(a) or II_(b) includes thefollowing structures:

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaIII

wherein

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹;-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   R¹⁰ is selected from —Y′—R¹⁸;-   Y′ is selected from a chemical bond, O, NR⁰—, and a hydrocarbon    chain having from 1 to 4 carbon atoms, and optionally substituted    with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, CO₂R⁰, C(O)R⁰, C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;-   R¹⁸ is selected from the group consisting of H, alkyl, cycloalkyl,    alkenyl, alkynyl, aralkyl, CF₃, aryl, and a heterocyclic ring; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

In a further preferred embodiment, the present invention providesinhibitors of the P210^(BCR-ABL-T315I) theramutein having the formulaIII_(a)

wherein:

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹;-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   X³ is N, CH or C—R⁰;-   each R⁵⁰ is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(r)C(O)(CH₂)_(s)R⁵¹, —(CH₂)_(r)C(O)N(R⁵²)(R⁵³),    —(CH₂)_(r)C(O)O(CH₂)_(s)R⁵¹, —(CH₂)_(r)N(R⁵¹)C(O)R⁵¹,    —(CH₂)_(r)N(R⁵²)(R⁵³), —N(R⁵¹)SO₂R⁵¹, —OC(O)N(R⁵²)(R⁵³),    —SO₂N(R⁵²)(R⁵³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R⁵⁰ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    -   R⁵¹ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   R⁵² and R⁵³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R⁵² and R⁵³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   r is 0 to 4;    -   s is 0 to 4;-   m is 0 to 4; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

Exemplary compounds of the formula III or III_(a) includes the followingstructures:

In a further embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula IV

wherein:

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹;-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   R²² is selected from H and C₁₋₃ alkyl;-   R³⁴ is selected from H, NO₂, CN, alkyl, cycloalkyl, alkenyl,    alkynyl, aralkyl, aryl and a heterocyclic ring;-   R⁴⁴ is selected from H, alkyl, cycloalkyl, —(C═O)R⁰, alkenyl,    alkynyl, aralkyl, aryl, and a heterocyclic ring;-   R⁴⁵ is selected from —Y″—R¹⁹;-   Y″ is selected from a chemical bond, O, NR⁰—, and a hydrocarbon    chain having from 1 to 4 carbon atoms, and optionally substituted    with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, CO₂R⁰, C(O)R⁰, C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;-   R¹⁹ is selected from the group consisting of H, alkyl, cycloalkyl,    alkenyl, alkynyl, aralkyl, CF₃, aryl, and a heterocyclic ring; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

Exemplary compounds of the formula IV include the following structures:

In a further embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula V

wherein:

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹.-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   R² is selected from H and C₁₋₃ alkyl;-   R³⁴ is selected from H, NO₂, CN, alkyl, cycloalkyl, alkenyl,    alkynyl, aralkyl, aryl and a heterocyclic ring;-   R⁵⁵ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, aryl, and a heterocyclic ring;-   R⁵⁶ is selected from —Y″—R¹⁹;-   Y″ is selected from a chemical bond, O, NR⁰—, and a hydrocarbon    chain having from 1 to 4 carbon atoms, and optionally substituted    with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, CO₂R⁰, C(O)R⁰, C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁴;-   R¹⁹ is selected from the group consisting of H, alkyl, cycloalkyl,    alkenyl, alkynyl, aralkyl, CF₃, aryl, and a heterocyclic ring; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

In a further embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula V_(a)

wherein:

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹.-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   R⁵⁵ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, aryl, and a heterocyclic ring;-   X³ is N or C—R⁵⁰;-   each R⁵⁰ is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁵¹,    —(CH₂)_(r)C(O)(CH₂)_(s)R⁵¹, —(CH₂)_(r)C(O)N(R⁵²)(R⁵¹),    —(CH₂)_(r)C(O)O(CH₂)_(s)R⁵¹, —(CH₂)_(r)N(R⁵¹)C(O)R⁵¹,    —(CH₂)_(r)N(R⁵²)(R⁵³), —N(R⁵¹)SO₂R⁵¹, —OC(O)N(R⁵²)(R⁵³),    —SO₂N(R⁵²)(R⁵³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R⁵⁰ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    -   R⁵¹ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   R⁵² and R⁵³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R⁵² and R⁵³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   r is 0 to 4;    -   s is 0 to 4;-   m is 0 to 4; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

Exemplary compounds of the formula V or V_(a) include the followingstructures:

In a further embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula VI

wherein:

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁴ or C—R¹.-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   R⁵⁵ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, aryl, and a heterocyclic ring;-   R⁵⁶ is selected from —Y″—R¹⁹;-   Y″ is selected from a chemical bond, O, NR⁰—, and a hydrocarbon    chain having from 1 to 4 carbon atoms, and optionally substituted    with one or more of halo, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, CO₂R⁰, C(O)R⁰, C(O)N(R⁰)₂, CN, CF₃, N(R⁰)₂, NO₂, and OR⁰;-   R¹⁹ is selected from the group consisting of H, alkyl, cycloalkyl,    alkenyl, alkynyl, aralkyl, CF₃, aryl, and a heterocyclic ring; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

In a further embodiment, the present invention provides inhibitors ofthe P210^(BCR-ABL-T315I) theramutein having the formula VI_(a)

wherein:

-   ring A is a 5-, 6-, or 7-membered ring or a 7- to 12-membered fused    bicyclic ring;-   X¹ is selected from N, N—R⁰ or C—R¹;-   X² is selected from N, N—R⁰ or C—R¹;-   the dotted lines represent optional double bonds;-   each R¹ is independently selected from the group consisting of H,    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR¹¹,    —(CH₂)_(p)C(O)(CH₂)_(q)R¹¹, —(CH₂)_(p)C(O)N(R¹²)(R¹³),    —(CH₂)_(p)C(O)O(CH₂)_(q)R¹¹, —(CH₂)_(p)N(R¹¹)C(O)R¹¹,    —(CH₂)_(p)N(R¹²)(R¹³), —N(R¹¹)SO₂R¹¹, —OC(O)N(R¹²)(R¹³),    —SO₂N(R¹²)(R¹³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R¹ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;-   n is 0 to 6,    -   each R¹¹ is independently selected from H, alkyl, cycloalkyl,        alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic ring;    -   each R¹² and R¹³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R¹² and R¹³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   p is 0 to 4;    -   q is 0 to 4;-   R⁵⁵ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,    aralkyl, aryl, and a heterocyclic ring;-   X³ is N or C—R⁵⁰;-   each R⁵⁰ is independently selected from the group consisting of    alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃, NO₂, OR⁵¹,    —(CH₂)_(r)C(O)(CH₂)_(s)R⁵¹, —(CH₂)_(r)C(O)N(R⁵²)(R⁵³),    —(CH₂)_(r)C(O)O(CH₂)_(s)R⁵¹, —(CH₂)_(r)N(R⁵¹)C(O)R⁵¹,    —(CH₂)_(r)N(R⁵²)(R⁵³), —N(R⁵)SO₂R⁵¹, —OC(O)N(R⁵²)(R⁵³),    —SO₂N(R⁵²)(R⁵³), halo, aryl, and a heterocyclic ring, and    additionally or alternatively, two R⁵⁰ groups on adjacent ring atoms    form a 5- or 6-membered fused ring which contains from 0 to 3    heteroatoms;    -   R⁵¹ is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl,        aralkyl, aryl, and a heterocyclic ring;    -   R⁵² and R⁵³ are independently selected from H, alkyl,        cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, and a heterocyclic        ring; or R⁵² and R⁵³ may be taken together with the nitrogen to        which they are attached form a 5- to 7-membered ring which may        optionally contain a further heteroatom;    -   r is 0 to 4;    -   s is 0 to 4;-   m is 0 to 4; and-   each R⁰ is independently selected from H, alkyl, cycloalkyl,    aralkyl, aryl and a heterocyclic ring.

Exemplary compounds of the formula VI or VI, include the followingstructures:

As used herein, the definition of each expression, e.g. alkyl, m, n, R,R′ etc., when it occurs more than once in any structure, is intended tobe independent of its definition elsewhere in the same structure.

For each of the above descriptions of compounds of the structures I,I_(a), I_(b), II, II_(a), II_(b), III, III_(a), IV, IV_(a), V, V_(a),VI, and VI_(a) each recitation of the terms halo, alkyl, cycloalkyl,alkenyl, alkynyl, aralkyl, aryl, heterocyclic group or heterocyclicring, are independently selected from the definitions of these terms asprovided in the beginning of this section.

It will be understood that chemical structures provided herein includethe implicit proviso that substitution is in accordance with permittedvalence of the substituted atom and the substituent(s), and that thesubstitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

When one or more chiral centers are present in the compounds of thepresent invention, the individual isomers and mixtures thereof (e.g.,racemates, etc.) are intended to be encompassed by the formulae depictedherein.

When one or more double bonds are present in the compounds of thepresent invention, both the cis- and trans-isomers are intended to beencompassed by the formulae depicted herein. Although chemicalstructures (such as, for example, structures II, II_(a), V, V_(a), VI,and VI_(a)) are depicted herein in either cis of trans configuration,both configurations are meant to be encompassed by the each of theformulae.

In certain embodiments, compounds of the invention may exist in severaltautomeric forms. Accordingly, the chemical structures depicted hereinencompass all possible tautomeric forms of the illustrated compounds.

The compounds of the invention may generally be prepared fromcommercially available starting materials and known chemical techniques.Embodiments of the invention may be synthesized as follows. One of skillin the art of medicinal or synthetic chemistry would be readily familiarwith the procedures and techniques necessary to accomplish the syntheticapproaches given below.

Embodiments wherein R²=NH, R³=N, R⁴=CH, and R⁵=-aryl may be prepared byreaction of an appropriate hydrazine compound, such as A, and anappropriate aldehyde, such as B, under conditions similar to thosedescribed on p. 562 of Gineinah, et al. (Arch. Pharm. Pharm. Med. Chem.2002, 335, 556-562).

For example, heating A with 1.1 equivalents of B for 1 to 24 hours in aprotic solvent such as a C₁ to C₆ alcohol, followed by cooling andcollection of the precipitate, would afford C. Alternatively, product Cmay be isolated by evaporation of the solvent and purification bychromatography using silica gel, alumina, or C₄ to C₁₈ reverse phasemedium. Similar methodology would be applicable in the cases where“Aryl” is replaced by other groups defined under R⁵.

Embodiments wherein R²=NH, R³=NR², R⁴=C(O), and R⁵=a heterocyclic ringmay be prepared by reaction of an appropriate hydrazine compound, suchas D, and an activated carboxylic acid such as E, wherein LG is aleaving group such as halo, 1-oxybenztriazole, pentafluorophenoxy,p-nitrophenoxy, or the like, or Compound E may also be a symmetricalcarboxylic acid anhydride, whereby conditions similar to those describedon p. 408 of Nair and Mehta (Indian J. Chem. 1967 5, 403-408) may beused.

For example, treatment of D with an active ester such asHeterocycle-C(O)—OC₆F₅ in an inert solvent such as dichloromethane,1,2-dichloroethane, or N,N-dimethylformamide, optionally in the presenceof a base such as pyridine or another tertiary amine, and optionally inthe presence of a catalyst such as 4-N,N-dimethylaminopyridine, at anappropriate temperature ranging from 0° C. to the boiling point of thesolvent, would afford F, which may be isolated by evaporation of thesolvent followed by chromatography using silica gel, alumina, or C₄ toC₁₈ reverse phase medium. The above active ester example of E would bereadily prepared from the corresponding carboxylic acid andpentafluorophenol using a carbodiimide such as dicyclohexylcarbodiimideas a condensing agent. Similar methodology would be applicable in thecases where “Heterocycle” is replaced by other groups defined under R⁵.

Precursors such as A and D may be prepared by reaction of an appropriatenucleophile, for example, a hydrazine derivative, with a heteroaromaticcompound bearing a halo substituent at a position adjacent to a nitrogenatom. For example, using methods analogous to those described by Wu, etal. (J. Heterocyclic Chem. 1990, 27, 1559-1563), Breshears, et al. (J.Am. Chem. Soc. 1959, 81, 3789-3792), or Gineinah, et al. (Arch. Pharm.Pharm. Med. Chem. 2002, 335, 556-562), examples of compounds A and D maybe prepared starting from, for example, a 2,4-dihalopyrimidinederivative, many of which are commercially available or are otherwisereadily prepared by one skilled in the art. Thus, treatment of anappropriate 2,4-dihalopyrimidine derivative G with an amine or othernucleophile (Z), optionally in the presence of an added base,selectively displaces the 4-halo substituent on the pyrimidine ring.Subsequent treatment of the product with a second nucleophilic reagentsuch as hydrazine or a hydrazine derivative, optionally in a solventsuch as a C₁ to C₆ alcohol and optionally in the presence of an addedbase, displaces the 2-halo substituent on the pyrimidine ring, to affordcompounds that are examples of structures A and D above.

Embodiments wherein R² is —NR²² and R³ is —C(═R³³) can be synthesized bymethods such as the following, or straightforward modifications thereof.The synthesis may be conducted starting from an appropriate ring Aderivative J that bears a leaving group (LG) adjacent to the requisitering nitrogen. Structure G above and the product of reaction ofstructure G with nucleophile Z, as illustrated above, are examples ofsuch appropriate Ring A derivatives J. Suitable LG′ groups are halo,alkylthio, alkylsulfonyl, alkylsulfonate or arylsulfonate. Treatment ofJ with an amine R¹²NH₂ effects displacement of LG′ to affordintermediates K. An example of this chemical transformation wherein R¹²is H and LG′ is CH₃SO₂— is reported by Capps, et al. J. Agric. FoodChem. 1993, 41, 2411-2415, and an example wherein R¹² is H and LG is Clis reported in Marshall, et al. J. Chem. Soc. 1951, 1004-1015.

Intermediates of structure K are transformed to compounds of theinvention by simultaneous or sequential introduction of the elements, ofR³, R⁴, and R⁵. For example, treatment of intermediates of structure Kwith individual isocyanates R⁶—N═C═O affords in a single step compoundsof structure M, which are compounds of the invention wherein R²=—NR²²—,R³=—C═O—, R⁴=—NH—, and R⁵=-chemical bond-R⁶. Alternative methods toconvert compounds of structure K to compounds of structure M are wellknown to those skilled in the art, wherein R³ together with a leavinggroup (for example p-nitrophenoxy or chloro) is first introduced,followed by subsequent displacement of the leaving group by, forexample, an amine R⁶—NH₂, to introduce R⁵ and R⁶.

Alternatively, treatment of intermediates of structure K with a reagentsuch as cyanamide (NH₂—CN), typically under conditions of heating andoptionally in the presence of acid in a solvent such as ethyl acetate ordioxane, affords intermediates N. Alternatives to cyanamide arenitroguanidine or amidinosulfonic acid (NH₂—C(═NH)—SO₃H). An example ofsuch a transformation using cyanamide is reported by Latham et al., J.Org. Chem. 1950, 15, 884. An example using nitroguanidine is reported byDavis, Proc. Natl. Acad. Sci. USA 1925, 11, 72. Use of amidinosulfonicacid was reported by Shearer, et al. Bioorg. Med. Chem. Lett. 1997, 7,1763.

In analogy to the conversion of intermediates A or D to embodimentsrepresented by C or F, intermediates K are converted, respectively, tocompounds represented by P or Q, which are further embodiments of theinvention.

Treatment of A or K with a ketone S, wherein R is as defined above, inplace of an aldehyde B in the schemes above, affords compounds ofstructure T or U, respectively, which are further embodiments of theinvention.

The non-guanidino carbon-nitrogen double bond of U can be selectivelyreduced by an appropriate reducing agent such as a metal (boron,aluminum, silicon, etc.) hydride reagents, preferably one with basicproperties, to afford compounds V of the invention.

Embodiments of the invention wherein R²=CO, R³=—NR²—, R⁴=N—, and R⁵=ZR⁷,wherein Z is a hydrocarbon chain and R⁷ is as defined above, may beprepared as follows. When R³²=H, a Ring A-derived carboxylic acid W isactivated by conversion to the corresponding acid chloride, oralternatively to an active ester, or to an analogous activatedderivative, many of which are well known in the art. Treatment of theactivated carboxylic acid with hydrazine affords the correspondinghydrazide Y. Treatment of Y with an aldehyde or ketone (under conditionsof heating and/or mild acid catalysis if necessary) affords the desiredfinal product Z.

If not commercially available, Ring A-derived carboxylic acids W may beprepared by treatment of starting material J above with cyanide ion,optionally with heating or transition metal catalysis, to replace theleaving group LG′ with a cyano residue. Basic or acidic hydrolysis ofthe cyano group affords the desired carboxylic acid intermediate W.

When R³² is not H, then a protected form of monosubstituted hydrazinemay be used in the above scheme in place of hydrazine. Thus, treatmentof the activated carboxylic acid from W with R³²NHNH-PG, where PG is anitrogen protecting group such as benzyloxycarbonyl ort-butyloxycarbonyl, followed by deprotection and treatment with anappropriate aldehyde or ketone as above affords Z′, a further embodimentof the invention.

It will be apparent to a practitioner skilled in the art of organicmolecule synthesis that the reaction processes illustrated above arerepresentative of a broader set of methods that are logical extensionsof the illustrated processes. Thus, additional embodiments of theinvention that incorporate additional variants in R², R³, R⁴, and R⁵claimed by this invention are prepared by obvious modifications of theabove processes.

As would be recognized by a person of ordinary skill, it may beadvantageous to employ a temporary protecting group in achieving thefinal product. The phrase “protecting group” as used herein meanstemporary modifications of a potentially reactive functional group whichprotect it from undesired chemical transformations. Examples of suchprotecting groups include esters of carboxylic acids, silyl ethers ofalcohols, and acetals and ketals of aldehydes and ketones, respectively.The field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P. G. M. Protective Groups in Organic Synthesis, 2^(nd) ed.;Wiley: New York, 1991).

A “mutein” is a protein having an amino acid sequence that is altered asa result of a mutation that has occurred in its corresponding gene(Weigel et al, 1989). Such mutations may result in changes in one ormore of the characteristics of the encoded protein. For example, anenzyme variant that has modified catalytic activity resulting from achange in one or more amino acids is a mutein.

This invention is concerned with proteins harboring an alteration of atleast one amino acid residue (the terms “amino acid sequence change” or“amino acid sequence alteration” include changes, deletions, oradditions, of at least one amino acid residue, or any combination ofdeletions, additions, changes) such that the resulting mutein has become(as a result of the mutation) resistant to a known therapeutic agentrelative to the sensitivity of the non-mutated version of said proteinto the therapeutic agent. This specialized class of muteins ishereinafter referred to as a theramutein, and the corresponding proteinlacking the mutation is referred to herein as a prototheramutein.

As used herein, “prototheramutein” refers to an endogenously occurringprotein in a cell that is susceptible to mutation that confers relativeinsensitivity (i.e. resistance) to a therapeutic compound whichotherwise inhibits or activates the protein. Accordingly, “theramutein”refers to an endogenously occurring protein or portion of a protein in acell that contains at least one amino acid sequence alteration relativeto an endogenous form of the protein, wherein the amino acid sequencechange is or was identified or becomes identifiable, and is or has beenshown to be clinically significant for the development or progression ofa given disease, following exposure of at least one human being to asubstance that is known to inhibit or activate the prototheramutein.Solely for the purposes of defining the preceding sentence, a substanceneed not be limited to a chemical agent for the purposes of firstdefining the existence of a theramutein. Thus, by definition, atheramutein is a protein which harbors a mutation in its correspondingendogenous gene, wherein said mutation is associated with thedevelopment of clinical resistance in a patient to a drug that isnormally able to activate or inhibit the non-mutated protein. Withrespect to a given theramutein, the term “correspondingprototheramutein” refers to the prototheramutein which, throughmutation, gives rise to said theramutein. Similarly, with respect to agiven prototheramutein, the “corresponding theramutein” refers to thetheramutein which has arisen by mutation from said prototheramutein.

Accordingly, it is apparent to a skilled artisan that, as the geneswhich encode theramuteins are limited to endogenously occurring genes,the definition of a theramutein excludes proteins encoded bydisease-causing infectious agents such as viruses and bacteria. As usedherein, the term “endogenous gene” refers to a gene that has beenpresent in the chromosomes of the organism at least in its unmutatedform, since inception. The term “cell” as used herein refers to a livingeukaryotic cell whether in an organism or maintained under appropriatelaboratory tissue or organ culture conditions outside of an organism.

In one aspect of the invention, a theramutein is a protein that isaltered for the first time with respect to a commonly occurring “wildtype” form of the protein (i.e. the prototheramutein). In another aspectof the invention, a theramutein is a variant of a protein(prototheramutein) that is, itself, already a mutein. In still anotherembodiment, a theramutein may be further mutated as compared to apreviously existing theramutein. In such instances, the firsttheramutein (such as the T315I mutant of p210 BCR-ABL (see below), maybe thought of as a “primary” theramutein, whereas subsequent mutationsof the (already mutated) T315I variant may be termed a secondarytheramutein, tertiary theramutein, etc. As exemplified below, a muteinof the invention is a variant of Bcr-Abl tyrosine kinase that escapesinhibition by an inhibitor of the “wild type” Bcr-Abl. Such a Bcr-Ablmutein is altered with respect to a more common or “wild type” form ofBcr-Abl (which is also a mutein as well) in such a way that a propertyof the protein is altered.

It will be understood that a mutein of primary interest is a theramuteinthat may have the same, increased, or decreased specific activityrelative to its prototheramutein, and that it is not inhibited or ispoorly inhibited by an agent that is capable of inhibiting theprototheramutein. Likewise, another theramutein of primary interest isone that has the same, increased or decreased specific activity(relative to its prototheramutein) and that is not activated or ispoorly activated by an agent that is capable of activating theprototheramutein. Other variations are obvious to the skilled artisan.It will be further appreciated that theramuteins can include naturallyoccurring or commonly observed variants of a protein, for example,variants that are expressed from different alleles of a particular gene.In some cases such variants may be unremarkable with respect to theirnormal cellular function, with functional differences becoming apparentonly in the presence of agents that differentially inhibit or activatethe cellular function of the variants. For example, naturally occurringvariants of a particular enzyme may have activity profiles that are notsubstantially different, but a therapeutic agent that modulates one maybe ineffective in modulating the other.

It will be appreciated that, whereas one aspect of the invention is theidentification of an agent that is active against a theramutein thatarises or becomes dominant (by any mechanism) during the course of atreatment for a given disease, another aspect is the identification ofan agent that is active against a mutein that is common within apopulation of unafflicted individuals, but wherein said mutein is lesssusceptible to modulation by an approved drug, and where the variationin the activity profile of the mutein becomes important (and istherefore first identified as being a theramutein) in a disease statesuch as where it is overexpressed or participates in a signaling processwhich has otherwise become abnormally regulated. For example, aneoplastic disease may be caused by abnormal regulation of a cellularcomponent other than the theramutein or its prototheramutein, and stillbe treatable with an inhibitor of the prototheramutein, whereas the sametreatment would be less effective or ineffective where the theramuteinwas present. This can be an issue where it is observed that the responseof a particular tumor type to an anticancer agent varies amongindividuals that express different variants of an enzyme against whichthe anticancer agent is directed (Lynch et al., 2004). Here, thevariants would not have arisen or become predominant during the courseof treatment of the disease, but are preexisting in the healthypopulation and are detected only by their altered responsiveness to aparticular course of established therapeutic treatment.

As used herein, the terms “agonist” and “activator” of a protein areused interchangeably. An activator (agonist) is limited to a substancethat binds to and activates the functioning of a given protein. Unlessexplicitly stated otherwise, an “activator”, an “agonist”, and an“activator of a protein” are identical in meaning. The activation by anactivator may be partial or complete. Likewise, as used herein, theterms “antagonist” and “inhibitor” of a protein are usedinterchangeably. An inhibitor (antagonist) is limited to a substancethat binds to and inhibits the functioning of a given protein. To statethat a substance “inhibit(s)” a protein means the substance binds to theprotein and reduce(s) the protein's activity in the cell withoutmaterially reducing the amount of the protein in the cell. Similarly, tostate that a substance “activate(s)” a protein, such as aprototheramutein or theramutein, is to state that the substanceincreased the defined function of the protein in the cell withoutsubstantially altering the level of the protein in the cell. Unlessexplicitly stated otherwise, an “inhibitor”, an “antagonist” and an“inhibitor of a protein” are also synonymous. The inhibition by aninhibitor may be partial or complete. A modulator is an activator or aninhibitor. By way of example, an “activator of PKC_(β1)” should beconstrued to mean a substance that binds to and activates PKC_(β1).Similarly, an “inhibitor of p210 Br-Ab” is a substance that binds to andinhibits the functioning of p210^(Bcr-Abl). To state that a substance“inhibits a protein” requires that the substance bind to the protein inorder to exert its inhibitory effect. Similarly, to state that asubstance “activates protein X” is to state that the substance binds toand activates protein X. The terms “bind(s),” “binding,” and “binds to”have their ordinary meanings in the field of biochemistry in terms ofdescribing the interaction between two substances (e.g.,enzyme-substrate, protein-DNA, receptor-ligand, etc.). As used herein,the term “binds to” is synonymous with “interacts with” in the contextof discussing the relationship between a substance and its correspondingtarget protein. As used herein, to state that a substance “acts on” aprotein, “affects” a protein, “exerts its effect on” a protein, etc.,and all such related terms uniformly mean (as the skilled investigatoris well aware) that said substance activates or inhibits said protein.

The concept of inhibition or activation of a mutated form of anendogenous protein to a greater extent than the correspondingnon-mutated counterpart protein is defined for the first time andreferred to herein as a positive “specificity gap.” In general terms,and using an inhibitor case as an example, the specificity gap refers tothe difference between the ability of a given substance, undercomparable conditions to inhibit the theramutein in a cell-based assaysystem as compared to either:

a) the ability of the same substance under comparable conditions toinhibit the prototheramutein, or

b) the ability of a second substance (usually a known inhibitor of theprototheramutein) to inhibit the theramutein under comparableconditions, or

c) the ability of the second substance to inhibit the prototheramuteinunder comparable conditions.

When the comparison is made between the effects of two distinctsubstances (tested individually) on the theramutein alone, the result istermed a homologous specificity gap determination.

Alternatively, when a comparison is made between the effects of twodistinct substances (generally, but not always), one of which is testedon the theramutein and the other on the prototheramutein, respectively,the result is termed a heterologous specificity gap (SG) determination.Thus, (a) and (c) as given above are examples of heterologousspecificity gap (SG) determinations (although (a) uses the samesubstance in both instances), whereas (b) is an example of a homologousspecificity gap determination.

Reference to FIG. 3 is informative in understanding and elucidatingthese concepts.

Analogous issues apply when the case concerns an activator. It will beimmediately obvious to the skilled artisan that the term “comparableconditions” includes testing two different compounds, for example, atthe same concentration (such as comparing two closely related compoundsto determine relative potency), or by comparing the effects of twodifferent compounds tested at their respective IC₅₀ values on thecorresponding prototheramutein and theramutein. The skilled investigatorwill easily recognize other useful variations and comparable conditions.

Thus, in one embodiment of the application of this approach, substancesthat are more effective against a theramutein have a “positivespecificity gap.” A “zero, null or no” specificity gap indicates thatthere is no significant measurable difference between the effect of asubstance on the theramutein as compared to its effect on theprototheramutein (however such compounds may be quite useful in theirability to inhibit or activate both a theramutein and its correspondingprototheramutein), and a “negative specificity gap” indicates asubstance that at a given concentration is less effective against thegiven theramutein than against a form of the correspondingprototheramutein or other comparative form of the theramutein (such asone that may harbor a different mutation). The latter category isgenerally of lesser interest than the former categories of compounds,except in the case where the compound is so potent that its relativelylesser effect on the theramutein is of no real concern from theperspective of therapeutic efficacy. The skilled investigator can easilyrecognize a variety of approaches to quantifying the specificity gapassessment in a manner tailored to his or her needs.

The invention also provides a means for identifying compounds thatexhibit a desired specificity gap. Such compounds can be identified andtheir ability to inhibit or activate the theramutein determined using anin vitro cell-based assay system where the effect of a substance on thecellular functioning of the mutated endogenous form of the protein iscompared to the effect of the same drug on the cellular functioning of anon-mutated endogenous form of the protein.

Thus, the system enables the discovery of compounds capable of bindingto a theramutein and exerting a greater modulatory effect on thecellular functioning of said theramutein than on its correspondingprototheramutein. Further, the system enables the discovery of compoundscapable of binding to a theramutein and exerting at least as great orgreater modulatory effect on the cellular functioning of a theramuteinthan previously known compounds are able to exert on the correspondingprototheramutein. In a particular embodiment of the invention, acompound may be screened for and identified that 1) is at least aseffective against the theramutein as the original drug is against theprototheramutein, and/or 2) is similarly effective against theprototheramutein as against the theramutein (i.e., displays a small oressentially zero specificity gap).

In an embodiment of the invention, cells that overexpress a theramuteinof interest are used to identify chemical agents that are inhibitors oractivators of (i.e., that bind to and inhibit or that bind to andactivate) at least the selected theramutein. The chemical agents mayalso be inhibitors or activators of the prototheramutein or even othertheramuteins of the same prototheramutein. As used herein, the terms“chemical agent” and “compound” are used interchangeably, and both termsrefer exclusively to substances that have a molecular weight up to, butnot including, 2000 atomic mass units (Daltons). Such substances aresometimes referred to as “small molecules.” Unless otherwise statedherein, the term substance as used herein refers exclusively to chemicalagents/compounds, and does not refer to biological agents. As usedherein, “biological agents,” are molecules which include proteins,polypeptides, and nucleic acids, and have molecular weights equal to orgreater than 2000 atomic mass units (Daltons).

According to the invention, a theramutein is selected and used in acell-based assay system designed to identify agents that are inhibitorsor activators of the theramutein. Where two or more distincttheramuteins originating from the same prototheramutein are known, it ispreferable to select the most resistant theramutein available for use inthe assay system. In general, the degree of resistance of a theramuteinto a given chemical agent is determined relative to its non-mutatedcounterpart (prototheramutein) using the drug that was firstadministered and known to inhibit or activate the prototheramutein andagainst which the theramutein “arose.” The methods of determining thedegree of such resistance, for example by analysis of IC₅₀ or AC₅₀values, are well known and standard in the art and will not bereiterated herein. However, no causal relationship is necessary orshould be inferred between the treatment of the patient with a giventherapeutic agent per se and the subsequent appearance of a theramutein.Rather, what is required in order to practice the invention is that atrue theramutein be properly selected according to the teachings herein.

Thus, for example, randomly generated site directed mutants of knownproteins that are created in the laboratory but that have not been shownto be clinically relevant are not appropriate muteins for use within thescope of this invention. Such muteins would not, of course, be properlyclassified as theramuteins.

For example, in an effort to obtain potential inhibitors of mutants ofp210^(Bcr-Abl), Huron et al. (2003) used a recombinant c-abl preparationand screened a series of compounds known to inhibit c-src tyrosinekinase activity. The authors performed c-abl kinase assays on theircompounds and identified the most potent compound as an 8 nM inhibitoragainst c-abl. When this compound (PD166326) was tested against variousp210^(Bcr-Abl) theramuteins, however, it showed activity against some ofthe mutants such as p210^(Bcr-Abl-E255K), but the p210^(Bcr-Abl-T315I)theramutein was found to remain 10 fold more resistant (Huron et al.2003, Table 3). Furthermore, in each case the compound was stillmarkedly less effective on the p210^(Bcr-Abl) theramuteins than it wasagainst the wild-type p210^(Bcr-Abl). When the compound was testedagainst p210^(Bcr-Abl-T315I) mutant activity, it was unable to inhibitthe activity to any appreciable extent (p. 1270, left hand column,second paragraph; see also FIG. 4.). Thus, the disclosed compound wasable to inhibit a theramutein that is partially resistant to STI-571,but had no activity against the T315I mutant of Bcr-Abl, which wasalready known at that time to be the theramutein that exhibited the mostresistance to STI-571. Hence purely and simply, the Huron methodologyfailed to identify an effective inhibitor of the p210^(Bcr-AblT315I)theramutein.

Indeed, prior to the disclosure of this invention, including both thedetailed methodology described for the first time herein as well as thecompositions provided herein, no one anywhere in the world has beensuccessful in identifying a chemical agent, let alone a methodology thatis capable of identifying a chemical agent that effectively inhibits thep210^(Bcr-AblT315I) theramutein to an equal or greater extent thanSTI-571 is able to do with respect to the wild type p210^(Bcr-Abl)protein. (See Shah et al., Science, July, 2004; O'Hare et al., Blood,2004; Tipping et al., Leukemia, 2004; Weisberg et al., Leukemia, 2004).

It cannot be overemphasized that such compounds would be immenselyuseful, because at the present time there is no alternative for patientswho progress to p210^(Bcr-Abl-T315I) theramutein-mediated imatinibmesylate-resistant status. Once patients develop such resistance, thereis no other effective alternative treatment available, and death iscertain. The method described herein provides the first reportedapproach to identify, pharmacologically characterize and chemicallysynthesize effective inhibitors of the p210^(Bcr-Abl-T315I) theramutein.Moreover, the skilled investigator will immediately recognize theapplicability and generalizability of this approach to any highlydrug-resistant theramutein.

In the present invention, a test cell is used that displays a carefullyselected phenotypic characteristic (as defined below) which is linked tothe presence and functional activity of the particulartheramutein-of-interest (TOI) in the cell under appropriate conditions.This should be qualitatively the same as the phenotypic characteristicdisplayed by a cell that expresses the prototheramutein. A phenotypiccharacteristic (i.e. a non-genotypic characteristic of the cell) is aproperty which is observed (measured), selected and/or defined forsubsequent use in an assay method as described herein. Expression of thephenotypic characteristic is responsive to the total activity of thetheramutein in the cell, and is a result of the absolute amount of thetheramutein and its specific activity. Often, the phenotypiccharacteristic is observable as a result of elevated levels oftheramutein activity and is not apparent in cells that express lowamounts of the theramutein or low amounts of its correspondingprototheramutein. Further, it can often be demonstrated that thephenotypic characteristic is modulated by modulating the specificactivity of the theramutein with an inhibitor or activator of thetheramutein, although this is not always the case since an inhibitor oractivator of the TOI may not always be available at the time the skilledinvestigator undertakes such a project. Thus, for the purpose ofdefining the phenotypic characteristic to be subsequently used with agiven test cell for assay purposes, the skilled investigator may alsouse a substance capable of increasing or decreasing the expression ofthe theragene, which will in turn lead to increases or decreases of thelevel of the corresponding theramutein. This allows the skilledinvestigator to simulate the effects of certain types of activators orinhibitors of the theramutein (such as a suicide inhibitor of thetheramutein, which is a class of chemical agent which binds irreversiblyand covalently modifies the TOI, rendering it permanently inactive),without actually having access to such a compound, for the purposes ofrefining the appropriate phenotypic characteristic for subsequentlyestablishing a useful cellular assay system. Examples known to one ofordinary skill that would be helpful for such purposes include the useof anti-sense DNA oligonucleotides, small interfering RNAs, other RNAinterference-based methodologies, and vector constructs containinginducible promoter systems. In this manner, the selected phenotypiccharacteristic is linked to the activity of the theramutein in the testcell. Notably for theramuteins, the selected phenotypic characteristicis usually also displayed by a cell that overexpresses theprototheramutein and in which the phenotypic characteristic is modulatedby known inhibitors or activators of the prototheramutein.

A phenotypic characteristic is simply a characteristic of a cell otherthan a genotypic characteristic of the cell. Except for the specificrequirements of a properly defined phenotypic characteristic asdisclosed herein for the purposes of creating useful cellular assaysystems according to the teachings of certain of the embodiments of theinvention, no other limitation of the term phenotypic characteristic ofany kind or nature is intended or appropriate in order to properly andeffectively practice the invention. Indeed, the skilled artisan must beable to select any characteristic of the cell that maximizes the utilityof establishing the proper cell-based assay for his or her needs. Thephenotypic characteristic can be quantitative or qualitative and beobservable or measurable directly (e.g., observable with the naked eyeor with a microscope), but most commonly the characteristic is measuredindirectly using standard automated laboratory equipment and assayprocedures which are known to those of skill in the art. The term“observable” means that a characteristic may be measured or is otherwisedetectable under appropriate conditions by any means whatsoever,including the use of any type of laboratory instrumentation available.The term “detectable” is not the same as “detected”. A characteristicmay be detectable to a skilled artisan without being detected at anygiven time, depending upon how the investigator chooses to design theassay system. For example, in searching for activators of aprototheramutein (or theramutein), it may be desirable to have therelevant phenotypic characteristic detected only after the addition of aknown activator or test substance capable of activating the POI. Thisprovides the ability to maximize the intensity of the signal that isgenerated by the test cell in the assay.

Phenotypic characteristics include but are not limited to growthcharacteristics, transformation state, differentiation state, substratephosphorylation state, catalytic activity, ion flux across the cellmembrane (calcium, sodium, chloride, potassium, hydrogen ions, etc.), pHchanges, fluctuations of second messenger molecules or otherintracellular chemical species such as cAMP, phosphoinositides, cyclicnucleotides, modulations of gene expression, and the like. Thecharacteristic of the cell may be observable or measurable continuously(e.g., growth rate of a cell), or after a period of time (e.g., terminaldensity of a cell culture), or transiently (e.g., modulation of a muteincauses a transient change in phosphorylation of a substrate of themutein, or a transient flux in ion flow across the membrane, orelevations or reductions in intracellular cAMP levels). In certainembodiments, a selected phenotypic characteristic may be detected onlyin the presence of a modulator of the prototheramutein or thetheramutein. No limitations are intended with respect to acharacteristic that may be selected for measurement. As used herein, theterms “characteristic of a cell” and “phenotypic characteristic”, andsimply “characteristic”, when used to refer to the particular measurableproperty of the intact cell or a subcellular fraction of the cellfollowing the treatment of a test cell with a substance, are identical.For example, a phenotypic characteristic can be focus formation thatbecomes observable when a cell that over expresses a selected protein iscultured in the presence of an activator of the protein, or it may be atransient increase or decrease in the level of an intracellularmetabolite or ion, such as cAMP, calcium, sodium, chloride, potassium,lithium, phosphatidylinositol, cGMP, bicarbonate, etc. It is obvious toone of ordinary skill in the art that after a cell is exposed to a testsubstance, the characteristic so measured (assayed) may be determined ona sub-cellular fraction of the cell. However, the initial treatment ofthe cell with a substance, which thereby causes the substance to comeinto contact with the cell, must be performed on the intact cell, not asub-cellular fraction.

The characteristic selected for measurement within the cell must not bean intrinsic physical or chemical property of the theramutein orprototheramutein itself (such as the mere amount (mass) of the proteininside the cell), but rather must be a characteristic that results fromthe activity of the theramutein inside the cell, thus affecting acharacteristic of the cell which is distinct from the theramuteinitself, as discussed in detail above. For example, where the theramuteinis a protein kinase that is capable of undergoing autophosphorylation, aprocess whereby the enzyme is capable of catalyzing the phosphorylationof itself by transferring a terminal phosphate group from ATP ontoitself, it would NOT be appropriate to select the phosphorylation stateof the TOI as an appropriate phenotypic characteristic of the cell formeasurement. This is because such a characteristic does not reflect theactivity of the TOI on other cellular components. As the skilledinvestigator knows, autophosphorylation is not necessarily reflective ofthe activity of a protein kinase in a cell, since mutants of proteinkinases are known that retain enzymatic activity sufficient to undergoautophosphorylation, yet have lost the capability to engage in signaltransduction events within the cell. The classic paper by White et al.(1988) is both educational and noteworthy in this respect.

The term “responsive phenotypic characteristic” means a characteristicof the cell which is responsive to inhibitors or activators of a givenprotein (prototheramutein or theramutein). The term “known therapeuticagent” is defined as any agent that has been administered to a humanbeing for the treatment of a disease in a country of the world.

A useful phenotypic characteristic, as exemplified herein in associationwith p210^(Bcr-Abl) and theramuteins thereof, is disregulation of cellgrowth and proliferation. It is noted that the same or similar assay maybe appropriate for use with many different proteins of interest. Forexample, disregulations of growth, proliferation, and/or differentiationare common phenotypic characteristics that may result fromoverexpression of a variety of different cellular proteins. It is animportant teaching of this invention that by overexpressing a selectedprotein in order to cause the appearance of such a phenotypiccharacteristic, the characteristic becomes linked to the presence,amount, and specific activity of that selected protein under suitableconditions, and this linkage allows the skilled investigator to identifyinhibitors or activators of a theramutein of interest (TOI) as desired.Accordingly, the phenotypic characteristic is responsive to changes inthe level and/or specific activity of the selected protein. Such aresponsive phenotypic characteristic is referred to herein as a“phenoresponse.”

Though not always necessary, it will often be advantageous to employcells that express high levels of the theramutein, and to select aphenotypic characteristic that results from overexpression of thetheramutein. This is because phenotypic characteristics linked to thefunctioning of the theramutein generally become more distinguishable(easier to measure) as a theramutein is overexpressed to a greaterextent. Further, phenoresponses that are observed in response tomodulators of the theramutein are often amplified as the functionallevel of the theramutein is increased. Expressed another way, theselected phenoresponse observed in cells that overexpress thetheramutein is particularly sensitive to modulators of the theramutein.

Preferably, the theramutein is stably expressed in a test cell. Stableexpression results in a level of the theramutein in the cell thatremains relatively unchanged during the course of an assay. For example,stimulation or activation of a component of a signaling pathway may befollowed by a refractory period during which signaling is inhibited dueto down-regulation of the component. For theramuteins of the invention,such down-regulation is usually sufficiently overcome by artificiallyoverexpressing the theramutein. Expressed another way, the expression issufficiently maintained that changes in a phenotypic characteristic thatare observed during the course of an assay are due primarily toinhibition or activation of the theramutein, rather than a change in itslevel, even if down-modulation of the theramutein subsequently occurs.For these reasons, although stable expression of the theramutein ispreferred, transfection followed by transient expression of thetheramutein may be employed provided that the selected phenotypiccharacteristic is measurable and the duration of the assay system isshort relative to the progressive decline in the levels of thetransiently expressed theramutein which is to be expected in suchsystems over time. For these reasons, stably expressing cell lines arepreferred (U.S. Pat. No. 4,980,281).

A preferred drug screening method of the present invention involves thefollowing:

1) Identification of a theramutein for which a novel inhibitor oractivator is desired. Identification of an appropriate theramutein maybe performed using standard techniques (See, Gorre et al., Science,2001; see also PCT/US02/18729). Briefly, patients that have been given acourse of a therapeutically effective treatment using an activator orinhibitor of a known or suspected prototheramutein and have subsequentlyshown clinical signs and symptoms consistent with disease relapse areidentified, and cells or tissue samples derived from such patients areobtained. Using standard laboratory techniques such as RT-PCR, thesequence of the prototheramutein is determined and compared to thepreviously determined nucleic acid sequence of the knownprototheramutein gene or cDNA sequence. Mutations, if present, areidentified and are correlated with functional resistance of theprototheramutein's function either in cell-based or, more commonly,cell-free assay systems, again using standard methodology. Onceresistance-inducing mutations are confirmed, then said one or moreconfirmed mutants comprise a defined theramutein which may be used inthe subsequent methods as described herein.

2) Provision of a test cell that expresses a theramutein of interest anddisplays an observable (measurable) phenotypic characteristic which hasbeen previously shown to be responsive to inhibitors or activators ofthe theramutein or, more commonly, the corresponding prototheramutein.Said specific phenotypic characteristic that has been previously shownto be responsive to inhibitors or activators of thetheramutein-of-interest (TOI), and/or the prototheramutein-of-interest(pTOI) is defined herein for the first time as a “phenoresponse.” Oneembodiment of this invention is the definitive use of the phenoresponsefor the purpose of identifying compounds that are likely to beinhibitors or activators of the TOI. This may be accomplished throughthe use of a high-throughput screen using a cell line overproducing agiven TOI and for which an appropriate phenoresponse has been identifiedand characterized. Alternatively, one may utilize a high-throughputprimary screen using a more generic phenotypic characteristic of a cellline (that does not qualify as a phenoresponse according to theteachings herein) and then utilize a secondary screen according to theteachings herein to distinguish between compounds that are true positive“hits”, i.e. inhibitors or activators of the theramutein of interest,from false positive compounds that are not inhibitors or activators ofthe theramutein of interest. In one embodiment, a cell is selected thatnaturally expresses the theramutein such that a responsive phenotypiccharacteristic is present under suitable culture conditions which areobvious to one of ordinary skill in the art. In other embodiments, thetheramutein is overexpressed, in some instances in a host cell that doesnot otherwise express the theramutein at all. This usually involvesconstruction of an expression vector from which the theramutein can beintroduced into a suitable host cell and overexpressed using standardvector systems and methodology. (Gorre et al., 2001; Housey et al.,1988). In one embodiment, overexpression results in a level of thetheramutein that is at least about 3 times the amount of the proteinusually present in a cell. Alternatively, the amount is at least about10 times the amount usually present in a cell. In another embodiment,the amount is at least about 20 times or more preferably at least about50 times the amount usually present in a cell.

3) Provision of a control cell that expresses the prototheramuteincorresponding to the theramutein of interest. As some of the muteinsthat are described herein are also enzymes, they usually retaincatalytic activity, and therefore the control cell usually displayssubstantially the same phenotypic characteristic as the test cell. Thephenotypic characteristic need not be quantitatively alike in bothcells, however. For example, a mutation that leads to reactivation ofthe prototheramutein may also increase, decrease, or otherwise affectits specific activity with respect to one or more of its substrates inthe cell. As a result, it may exhibit the selected phenotypiccharacteristic to a greater or lesser extent. Accordingly, it may bedesirable in some cases to adjust expression of either or both of theprototheramutein and the theramutein such that test and control cellsexhibit the phenotypic characteristic to approximately the same degree.This may be done, for example, by expressing the proteins from promoterswhose activity can be adjusted by adjusting the amount of inducerpresent, all using standard methodology (see, for example, Sambrook etal. 1989 & 2001).

It will be obvious to one of ordinary skill in the art that a properlydefined phenoresponse may be quantitatively different between theprototheramutein- and the theramutein-expressing cell lines as a resultof differences in the specific activity (if any) between the theramuteinand its corresponding prototheramutein. Theramutein-inducing mutationsmay increase or decrease the specific activity of said theramuteinrelative to the corresponding prototheramutein. When comparing atheramutein expressing cell line with a prototheramutein expressing cellline, it is preferable that the selected phenoresponse is qualitativelythe same in both cell types. Thus, the skilled investigator may chooseto normalize the activity of the theramutein-expressing cell line tothat of the prototheramutein-expressing cell line, or vice versa. Suchnormalization methods are standard in the art. See, for example, Bolstadet al. (2003).

Alternatively, the skilled investigator may also wish to use unmodifiedhost cells or host cells harboring the expression vector only as controlcells for certain experimental procedures. (The host cells are the cellsinto which an expression vector encoding the theramutein was introducedin order to generate the test cells.) This may be the case where theinvestigator is only interested in identifying a specific inhibitor oractivator of the theramutein of interest, irrespective of whether or notsaid compound is also effective the prototheramutein of interest (pTOI).

4) The test and control cells are then maintained or propagated(although not necessarily at the same time) in growth media (or even inintact animals) under suitable conditions such that the phenoresponsemay be expressed and assayed. Control cells that are expressing theprototheramutein may be treated with a known modulator of theprototheramutein, or with a test substance, and test cells are treatedwith test compounds to determine whether they are active against thetheramutein, as measured by the ability of said substances to modulatethe phenoresponse in the expected manner. Alternatively, control cellsnot expressing the prototheramutein may also be substituted, dependingupon the particular phenoresponse that the skilled investigator haschosen for study. Substances may then be assayed on the test cells and,optionally, on the control cells at the same time, or at another time,and the results compared.

In one embodiment of the invention, substances that are active withregard to the test cells can be rapidly identified by their ability tomodulate the phenoresponse of the test cells in the same manner as, forexample, the known modulator of the prototheramutein alters thephenoresponse of prototheramutein-expressing control cells. In anotherembodiment, active substances may be identified by their ability tomodulate the activity of the theramutein in the test cells while havinglittle or no effect on the unmodified (prototheramutein and/ortheramutein non-expressing) control cells. The skilled investigator willreadily appreciate the many variations of this approach that may beutilized to identify, for example, modulators that are more effectiveagainst the theramutein, or that are equally effective against both theprototheramutein and one or more corresponding specific theramuteins.

Other phenoresponses can be observed and/or measured and include, forexample, detection of substrates of the prototheramutein, and detectionof gene expression changes that are regulated by the activity of thetheramutein. In the simplest terms, any characteristic of the cell thatthe skilled investigator has previously correlated with the functionalactivity of the theramutein may be suitable for use with such methods.However, in selecting a given characteristic, the skilled investigatormust first verify that said characteristic fulfills the criteria ofbeing a phenoresponse according the teachings as given in detail herein.The skilled investigator may also wish to normalize the phenoresponsewith the theramutein expressing cells to that of the prototheramuteinexpressing cells.

Characteristics suitable for detection may be measured by a variety ofmethods very well known to those of skill in the art. Such methodsinclude, but are not limited to, detection of fluorescence of suitablylabeled proteins (FACS), immunohistochemistry (IHC) for detection ofprotein expression, competitive radioligand binding assays, solid matrixblotting techniques, such as Northern, Southern, and Western blots ofcell extracts, reverse transcriptase polymerase chain reaction (RT-PCR),enzyme linked immunosorbent assays (ELISA), phosphorylation assays, gelretardation assays, membrane potential perturbations, and the like. Therelevant phenotypic characteristic may be detected either on the intactcell after treatment with a test substance or, alternatively, on asubcellular fraction of the cell after treatment of the intact cell witha test substance.

Once compounds are identified that have the desired effect on thetheramutein expressing test cells, it may be desirable (but notnecessary) to independently verify that the compounds identified areexerting their effects on the theramutein through a direct bindingmechanism, i.e. that the compounds fulfill the criteria of beinginhibitors or activators (as desired) of the theramutein according tothe teachings of the invention (the reader is referred to thedefinitions of the terms “activator” and “inhibitor” as given above).This may be accomplished with numerous standard binding assays that areknown to one of ordinary skill in the art, involving either purifiedprotein samples or intact cellular binding assays using cellstransfected with the appropriate prototheramutein or theramuteintogether with appropriate controls as dictated by sound scientificmethods. Since such methods are well established in the art they willnot be reiterated here. Numerous reference texts comprehensively discusssuch techniques (see, for example, Foreman and Johansen, 2002; Enna S.J. et al. (1991) Current Protocols in Pharmacology, Wiley & Sons,Incorporated; Bonifacino, J. S. et al. (1999) Current Protocols in CellBiology, Wiley & Sons, Incorporated). See also Housey, G. M. 1988,Chapter 4, and references therein; see also Horowitz et al., 1981.

In a particular embodiment of the invention, the method is used toidentify substances that are inhibitors of the p210^(Bcr-Abl-T315I)theramutein. The prototheramutein and theramutein are each expressed inBa/F3 (murine) cells using standard methodology and the phenoresponsesthat are observed are growth characteristics (terminal cell density fora carefully defined cell culture, and growth in the absence ofInterleukin-3 (IL-3). Unmodified host cells, or host cells containingthe expression vector only or both, may optionally also be used. Instill another embodiment, the test cells alone may be used with orwithout reference to a known inhibitor or activator.

Another useful assay is the determination of the state ofphosphorylation of a direct substrate of p210^(Bcr-Abl-T315I). One suchsubstrate is Crkl (Gorre et al., Science 293:876-80 (2001)), an adapterprotein which mediates the connection between Bcr-Abl and Ras. Thephosphorylation state of CRKL is representative of the signalingactivity of p210^(Bcr-Abl) in a cell. Another downstream substrate isp62DOK. Any such substrate would suffice for these purposes, provided ofcourse that phosphorylation of said substrate has been shown to occurinside the cell, and is not simply an autophosphorylation event of theTOI or PTOI as discussed above. Other signal transduction cascadecomponents may also be monitored, including src family kinases, STAT5,PI3 Kinase, raf kinase, RAS, MEK, ERK1 and ERK2, JNK1, 2 and 3, MLK1, 2and 3, MKK4, MKK7, AKT, mTOR, HSP90, and others.

As exemplified herein, inhibitors of the T315I theramutein have beenidentified. Furthermore, these inhibitors are also active to differingextents against the wild type prototheramutein p210^(Bcr-Abl-wt).

According to the present invention, a therapeutically effective amountof one or more compounds that modulate the functional activity of ap210^(Bcr-Abl) theramutein is administered to a mammal in need thereof.The term “administering” as used herein means delivering the compoundsof the present invention to a mammal by any method that may achieve theresult sought. They may be administered, for example, orally,parenterally (intravenously or intramuscularly), topically,transdermally or by inhalation. The term “mammal” as used herein isintended to include, but is not limited to, humans, laboratory animals,domestic pets and farm animals. “Therapeutically effective amount” meansan amount of a compound that, when administered to a mammal, iseffective in producing the desired therapeutic effect, such asinhibiting kinase activity, inhibiting cancer cell growth and division,etc.

The invention provides a method of treating disease in a mammal byadministering to the mammal an effective amount of a modulator of atheramutein. Suitable diseases to be treated according to the presentinvention include, but are not limited to, relapsing neoplastic or otherproliferative disorders that have become resistant to previouslyadministered drugs. The method is also useful for overcoming variationamong individuals with respect to susceptibility to drug treatment thatresults from allelic differences among therapy targets. For example, therole of p210^(Bcr-Abl) tyrosine kinase signaling in CML has beenextensively demonstrated, as has the role of theramuteins ofp210^(Bcr-Abl) in drug resistant recurrence of CML. Further, differentmuteins of p210^(Bcr-Abl) exhibit varying sensitivity to inhibitors ofp210^(Bcr-Abl). Although some theramuteins arise during drug therapy,others may preexist in the population. These latter examples will not berecognized as theramuteins until such time as the disease state ensuesand is followed by treatment with a known class of therapeutic agents.Only after said treatment will such preexisting theramuteins revealthemselves as being clinically significant in terms of relativenon-responsiveness leading to the progression of the disease in thepatient harboring the theramutein.

In an embodiment of the invention, theramutein modulators areadministered in combination with one or more other anti-neoplasticagents. Any suitable anti-neoplastic agent can be used, such as achemotherapeutic agent, radiation or combinations thereof. Theanti-neoplastic agent can be an alkylating agent or an anti-metabolite.Examples of alkylating agents include, but are not limited to,cisplatin, cyclophosphamide, melphalan, and dacarbazine. Examples ofanti-metabolites include, but not limited to, doxorubicin, daunorubicin,and paclitaxel, gemcitabine, and topoisomerase inhibitors irinotecan(CPT-11), aminocamptothecin, camptothecin, DX-8951f, topotecan(topoisomerase I inhibitor), and etoposide (VP-16; topoisomerase TTinhibitor) and teniposide (VM-26; topoisomerase II inhibitor). When theanti-neoplastic agent is radiation, the source of the radiation can beeither external (external beam radiation therapy—EBRT) or internal(brachytherapy—BT) to the patient being treated. The dose ofanti-neoplastic agent administered depends on numerous factors,including, for example, the type of agent, the type and severity of thetumor being treated and the route of administration of the agent. Itshould be emphasized, however, that the present invention is not limitedto any particular dose, route of administration, or combination ofchemotherapeutic agents or other therapeutic regimens that are combinedwith the administration of theramutein modulators.

Anti-neoplastic agents which are presently known in the art or beingevaluated can be grouped into a variety of classes including, forexample, mitotic inhibitors, alkylating agents, anti-metabolites,intercalating antibiotics, growth factor inhibitors, cell cycleinhibitors, enzymes, topoisomerase inhibitors, anti survival agents,biological response modifiers, anti-hormones, and anti-angiogenesisagents, all of which can be administered with inhibitors or activatorsof theramuteins.

A modulator of a theramutein can be administered with antibodies thatneutralize other receptors involved in tumor growth. Further, amodulator of a theramutein can be administered with a compound thatotherwise modulates a component of a signal transduction pathway,preferably a component of the signal transduction pathway in which thetheramutein is active and which is common to one or more other signaltransduction pathways. In an embodiment of the invention, a theramuteinmodulator is used in combination with a receptor antagonist that bindsspecifically to the Epidermal Growth Factor Receptor (EGFR).Particularly preferred are antigen-binding proteins that bind to theextracellular domain of EGFR and block binding of one or more of itsligands and/or neutralize ligand-induced activation of EGFR. An EGFRantagonist can be an antibody that binds to EGFR or a ligand of EGFR andinhibits binding of EGFR to its ligand. Ligands for EGFR include, forexample, EGF, TGF-α, amphiregulin, heparin-binding EGF (HB-EGF) andbetacellulin. EGF and TGF-α are thought to be the main endogenousligands that result in EGFR-mediated stimulation, although TGF-α hasbeen shown to be more potent in promoting angiogenesis. It should beappreciated that the EGFR antagonist can bind externally to theextracellular portion of EGFR, which can or can not inhibit binding ofthe ligand, or internally to the tyrosine kinase domain in the case ofchemical agents. Examples of EGFR antagonists that bind EGFR include,without limitation, biological agents such as antibodies (and functionalequivalents thereof) specific for EGFR, and chemical agents (smallmolecules), such as synthetic kinase inhibitors that act directly on thecytoplasmic domain of EGFR.

Other examples of growth factor receptors involved in tumorigenesis arethe receptors for vascular endothelial growth factor (VEGFR-1 andVEGFR-2), platelet-derived growth factor (PDGFR), nerve growth factor(NGFR), fibroblast growth factor (FGFR), and others.

In a combination therapy, the theramutein inhibitor is administeredbefore, during, or after commencing therapy with another agent, as wellas any combination thereof, i.e., before and during, before and after,during and after, or before, during and after commencing theanti-neoplastic agent therapy. For example, the theramutein inhibitorcan be administered between 1 and 30 days, preferably 3 and 20 days,more preferably between 5 and 12 days before commencing radiationtherapy. In a preferred embodiment of the invention, chemotherapy isadministered prior to, concurrently with or, more preferably, subsequentto antibody therapy.

In the present invention, any suitable method or route can be used toadminister theramutein inhibitors of the invention, and optionally, toco-administer anti-neoplastic agents and/or antagonists of otherreceptors. The anti-neoplastic agent regimens utilized according to theinvention, include any regimen believed to be optimally suitable for thetreatment of the patient's neoplastic condition. Different malignanciescan require use of specific anti-tumor antibodies and specificanti-neoplastic agents, which will be determined on a patient to patientbasis. Routes of administration include, for example, oral, intravenous,intraperitoneal, subcutaneous, or intramuscular administration. The doseof antagonist administered depends on numerous factors, including, forexample, the type of antagonists, the type and severity of the tumorbeing treated and the route of administration of the antagonists. Itshould be emphasized, however, that the present invention is not limitedto any particular method or route of administration.

Suitable carriers include, for example, one or more of water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, aswell as combinations thereof. Carriers can further comprise minoramounts of auxiliary substances, such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the theramutein modulator as the active ingredient. The compositionscan, as is well known in the art, be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the mammal.

The compositions of this invention can be in a variety of forms. Theseinclude, for example, solid, semi-solid and liquid dosage forms, such astablets, pills, powders, liquid solutions, dispersions or suspensions,liposomes, suppositories, injectable and infusible solutions. Thepreferred form depends on the intended mode of administration andtherapeutic application.

Such compositions of the present invention are prepared in a manner wellknown in the pharmaceutical art. In making the composition the activeingredient will usually be mixed with a carrier, or diluted by a carrierand/or enclosed within a carrier which can, for example, be in the formof a capsule, sachet, paper or other container. When the carrier servesas a diluent, it can be a solid, semi-solid, or liquid material, whichacts as a vehicle, excipient or medium for the active ingredient. Thus,the composition can be in the form of tablets, lozenges, sachets,cachets, elixirs, suspensions, aerosols (as a solid or in a liquidmedium), ointments containing, for example, up to 10% by weight of theactive compound, soft and hard gelatin capsules, suppositories,injection solutions, suspensions, sterile packaged powders and as atopical patch.

It should be appreciated that the methods and compositions of thepresent invention can be administered to any suitable mammal, such as arabbit, rat, or mouse. More preferably, the mammal is a human.

The compounds according to the invention may also be present as salts.In the context of the invention, preference is given to pharmaceuticallyacceptable salts. Pharmaceutically acceptable salts refers to an acidaddition salt or a basic addition salt of a compound of the invention inwhich the resulting counter ion is understood in the art to be generallyacceptable for pharmaceutical uses. Pharmaceutically acceptable saltscan be salts of the compounds according to the invention with inorganicor organic acids. Preference is given to salts with inorganic acids,such as, for example, hydrochloric acid, hydrobromic acid, phosphoricacid or sulfuric acid, or to salts with organic carboxylic or sulfonicacids, such as, for example, acetic acid, maleic acid, fumaric acid,malic acid, citric acid, tartaric acid, lactic acid, benzoic acid, ormethanesulfonic acid, ethanesulfonic acid, phenylsulfonic acid,toluenesulfonic acid or naphthalenedisulfonic acid. Pharmaceuticallyacceptable salts can also be metal or ammonium salts of the compoundsaccording to the invention. Particular preference is given to, forexample, sodium, potassium, magnesium or calcium salts, and also toammonium salts which are derived from ammonia or organic amines, suchas, for example, ethylamine, di- or triethylamine, di- ortriethanolamine, dicyclohexylamine, dimethylaminoethanol, arginine,lysine, ethylenediamine or 2-phenylethylamine. (see, Berge et al. J.Pharm. Sci. 1977, 66, 1-19).

-   1. Throughout this application, various publications, reference    texts, textbooks, technical manuals, patents, and patent    applications have been referred to. The teachings and disclosures of    these publications, patents, patent applications and other documents    in their entireties are hereby incorporated by reference into this    application to more fully describe the state of the art to which the    present invention pertains.-   2. It is to be understood and expected that variations in the    principles of invention herein disclosed may be made by one skilled    in the art and it is intended that such modifications are to be    included within the scope of the present invention.-   3. The following examples further illustrate the invention, but    should not be construed to limit the scope of the invention in any    way. Detailed descriptions of conventional methods, such as those    employed in the construction of vectors and plasmids, the insertion    of genes encoding polypeptides into such vectors and plasmids, the    introduction of plasmids into host cells, and the expression and    determination thereof of genes and gene products can be obtained    from numerous publications, including Sambrook, J et al., (1989)    Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring    Harbor Laboratory Press; Coligan, J. et al. (1994) Current Protocols    in Immunology, Wiley & Sons, Incorporated; Enna, S. J. et al. (1991)    Current Protocols in Pharmacology, Wiley & Sons, Bonifacino, J. S.    et al. (1999) Current Protocols in Cell Biology, Wiley & Sons, and    U.S. Pat. No. 4,980,281. All references mentioned herein are    incorporated in their entirety.

Examples

p210^(Bcr-Abl-T315I) is a theramutein of the p210Bcr-Abl protein(p210^(Bcr-Abl)) that is resistant to inhibition by imatinib mesylate(Gleevec, STI-571). The mutation at position 315 converts a threonine toan isoleucine residue and is one of several mutations that are observedamong resistant or relapsed patients. This particular mutant, however,is the most resistant such theramutein yet identified.

A phenoresponse was determined for a Ba/F3 cell line engineered tooverexpress the p210^(Bcr-Abl-T315I) theramutein. The phenoresponse wasdetermined relative to non-transformed Ba/F3 cells and Ba/F3 cells thatexpress the p210^(Bcr-Abl-wt) prototheramutein. The phenoresponse wasthe ability of the T315I mutants to grow to a higher cell saturationdensity under analogous culture conditions as compared to the controlnon-transformed Ba/F3 cell line, and to grow in the absence ofinterleukin 3 (IL-3), which is required for maintenance of the controlnon-transformed Ba/F3 cell line. The phenoresponse was defined andcharacterized according to the teachings given above.

The detection system utilized was a high speed cell imaging and countingsystem in which 3 μl sample volumes of cells were sequentially injectedthrough a 5 μl optical microcell, digitally imaged and electronicallystored, scanned, and then counted, all under a microcomputer-basedcontrol system. The system has the capacity to perform direct cellcounts on samples from cultures as small as 500 μl and providesstatistically significant total cell counts from culture samplescontaining as few as 12,500 cells. All of the figures displaying cellcount and viability assays utilized this system for data acquisition andanalysis. Simultaneously with the cell count performed, the system isalso capable of determining overall cell viability by distinguishingcounted, imaged cells that have excluded trypan blue (counted as“viable” cells) from cells which have taken up the trypan blue dye(counted as “non-viable” cells). Injection of trypan blue into the cellsample occurs immediately prior to the sample being sequentiallyinjected into the microcell for simultaneous cell counting and imaging.

The system may be integrated into the workflow of high-throughputscreening devices to provide a sensitive and precise cell counting andcell viability assay system that is more reliable and less prone toconfounding effects of metabolic viability-based cellular assays such asXTT or Alamar blue.

Initially, approximately 113,000 compounds were screened atconcentrations generally ranging from 10 to 20 μM to identify a subsetthat was capable of affecting growth of Ba/F3 cells (Ba/F3 T315I cells)overexpressing the p210^(Bcr-Abl-T315I) theramutein by any means.

A total of approximately 11,760 compounds showed greater than 50% growthinhibition, which were thought to correspond to approximately 4500distinct chemical classes. Retesting of these compounds with the samecell line yielded a database of compound responsiveness which was thensorted and rank ordered according to those compounds exhibiting thehighest overall growth inhibition. From this rank ordered database, thehighest scoring 130 compounds (based upon the greatest degree of growthinhibition observed at the lowest concentrations that compounds weretested) were then rescreened in a defined cell-based assay system usingBa/F3 T315I as test cells and wild type Ba/F3 as control cells accordingto the methods of the present invention. Compounds of interest werethose that differentially inhibited growth of Ba/F3 cells expressing thep210^(Bcr-Abl-T315I) theramutein relative to non-transformed wild typeBa/F3 cells. Six compounds were identified that fulfilled the desiredcriteria, and some of these compounds were analyzed in further detailusing the Ba/F3 p210^(Bcr-Abl-wt) cells line (Ba/F3 P210 cells) as well.One compound was unavailable for further testing due to lack ofavailability of additional material from the chemical supplier. Theremaining five compounds were independently evaluated in additionalcell-based assays using the aforementioned cell lines as well as in acell-free purified protein kinase assay using human recombinantlyproduced 120 Kd kinase domain fragments isolated from both wild typeP210 Bcr-Abl as well as P210 T315I mutant kinase domain.

All five compounds inhibited p210^(Bcr-Abl-T315I) 120 Kd activity asmeasured by inhibition of autophosphorylation activity, as shown in FIG.4. Thus, of the 6 highest scoring compounds out of more than 113,000compounds screened, at least 5 of the six directly inhibited thep210^(Bcr-Abl-T315I) mutant. It is noteworthy that Compound 5 appears tospread the recombinant protein band out on the SDS page gel. This wasalso evident on the silver-stained gel (data not shown). It is possiblethat this compound may actually be a “suicide” inhibitor that is able tocovalently cross-link the POI in order to permanently inhibit itsactivity, but this will require further study.

Taken together, the teachings and the results described herein provideconclusive proof that the system is capable of identifying inhibitors oractivators of the selected theramutein, and the skilled investigatorwill immediately recognize that such a system may be easily applied toany other theramutein with only obvious, minor modifications.

Representative examples of the cell-based assay results demonstratingselective inhibition of growth of the Ba/F3 T315I cell line relative tothe wild type non-transformed Ba/F3 cells are shown in FIGS. 1 and 2.The compounds inhibited growth and reduced the viability of cellsexpressing the T315I theramutein at concentrations under which thegrowth and viability of the wild type Ba/F3 non-transformed cells (notexpressing either p210^(Bcr-Abl-wt) or p210^(Bcr-Abl-T315I)) wererelatively unaffected, whereas cells expressing both theprototheramutein as well as the theramutein were substantiallyinhibited. In some instances, the T315I expressing cells were inhibitedto an even greater extent than the P210 prototheramutein expressingcells. (See, for example, FIG. 3, right hand side, Compound 3 resultsagainst P210 and T315I cells.

In summary, the methods presented herein provide a fundamental advancein the form of a generalizable approach for creating or identifyingmodulators of any given theramutein. The results demonstrateconclusively the power of the method to identify critically neededcompounds to overcome a specific type of acquired drug resistance thatis uniformly fatal in certain patient populations and is presentlyuntreatable. Furthermore, it is evident to one of skill in this art thatthe techniques and methods described herein may, using obviousmodifications, be straighforwardly generalized to any potentialtheramutein of clinical significance.

It is remarkable that out of a primary screen of more than 100,000compounds where approximately 10,000 compounds exhibited some degree ofgrowth inhibition, when the most potent growth inhibitory substanceswere rescreened using the Method described in detail herein, 6 distinctcompounds were identified and all of the compounds that weresubsequently tested exhibited inhibitory activity in a cell-freepurified protein kinase assay using the T315I mutant (one compound wasunavailable for further testing). Based upon such remarkable results, itbecomes immediately clear to the skilled artisan that the method may beeffectively applied toward the identification of inhibitors oractivators of an theramutein based upon the proper selection anddefinition of the phenoresponse according to the teachings in thesections given above and the documents incorporated by reference herein.For example, with knowledge of the foregoing, one of ordinary skill inthe art could easily design an assay system to identify inhibitors oftheramuteins derived from other prototheramuteins known to exhibitmutations that confer drug resistance such as the c-kit gene product orthe Epidermal Growth Factor (EGF) Receptor (EGFR), or the PlateletDerived Growth Factor (PDGF) Receptor a and 3. No limitation should beinferred upon the utility of the method with respect to its ability tobe utilized with any given theramutein expressed in any mammalian celltype for which a corresponding phenoresponse is detectable.

All references to any publication, patent, or other citation are herebyincorporated by reference.

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1.-53. (canceled)
 54. (canceled) 55.-64. (canceled)
 65. A method oftesting a compound, the method comprising: a) incubating a test cellwhich expresses a theramutein with a compound, the theramutein beingcapable of producing a detectable phenoresponse of the test cell; b)incubating a control cell which expresses a prototheramuteincorresponding to the theramutein with a known modulator of theprototheramutein, the prototheramutein being capable of producing thephenoresponse in the control cell; c) comparing the phenoresponse of thecontrol cell to the known modulator of the prototheramutein to thephenoresponse of the test cell to the compound; and d) determining thatthe compound is a modulator of the theramutein when the phenoresponse ofthe test cell is modulated to at least the same degree as thephenoresponse of the control cell is modulated by the known modulator ofthe prototheramutein.
 66. The method as recited in claim 65, wherein aspecificity gap, defined by a difference between the ability of thecompound to modulate the theramutein and the ability of the knownmodulator of the prototheramutein to modulate the prototheramutein, ispositive.
 67. The method as recited in claim 65, wherein either thetheramutein or its corresponding prototheramutein is a component of asignal transduction cascade.
 68. The method as recited in claim 65,wherein either the theramutein or its corresponding prototheramutein isa protein kinase.
 69. The method as recited in claim 65, wherein thephenoresponse of the test cell comprises at least one of a growthcharacteristic of the test cell, a transformation state of the testcell, and a differentiation state of the test cell.
 70. The method asrecited in claim 65, wherein the phenoresponse of the test cellcomprises at least one of a phosphorylation state of a second protein,that is not the theramutein, in the test cell, a change in ion fluxacross a membrane of the test cell, a change in pH within the test cell,and a change in concentration of an intracellular chemical specieswithin the test cell.
 71. The method as recited in claim 65, wherein thephenoresponse of the test cell comprises modulation of the geneticexpression of the theramutein within the test cell.